Compositions and methods for producing enhanced antibodies

ABSTRACT

The invention provides a Vitaxin antibody and a LM609 grafted antibody exhibiting selective binding affinity to α v β 3 . The Vitaxin antibody consists of at least one Vitaxin heavy chain polypeptide and at least one Vitaxin light chain polypeptide or functional fragments thereof. Also provided are the Vitaxin heavy and light chain polypeptides and functional fragments. The LM609 grafted antibody consists of at least one CDR grafted heavy chain polypeptide and at least one CDR grafted light chain polypeptide or functional fragment thereof. The invention additionally provides a high affinity LM609 grafted antibody comprising one or more CDRs having at least one amino acid substitution, where the α v β 3  binding activity of the high affinity LM609 grafted antibody is enhanced. Nucleic acids encoding Vitaxin and LM609 grafted heavy and light chains as well as nucleic acids encoding the parental non-human antibody LM609 are additionally provided. Functional fragments of such encoding nucleic acids are similarly provided. The invention also provides a method of inhibiting a function of α v β 3 . The method consists of contacting α v β 3  with Vitaxin or a LM609 grafted antibody or functional fragments thereof under conditions which allow binding to α v β 3 . Finally, the invention provides for a method of treating an α v β 3 -mediated disease. The method consists of administering an effective amount of Vitaxin or a LM609 grafted antibody or functional fragment thereof under conditions which allow binding to α v β 3 .

[0001] This application is a continuation-in-part of U.S. Ser. No.08/791,391, filed Jan. 30, 1997, the entire contents of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to integrin mediateddiseases and, more particularly, to nucleic acids encodingα_(v)β₃-inhibitory monoclonal antibodies and to CDR graftedα_(v)β₃-inhibitory antibodies for the therapeutic treatment ofα_(v)β₃-mediated diseases.

[0003] Integrins are a class of cell adhesion receptors that mediateboth cell-cell and cell-extracellular matrix adhesion events. Integrinsconsist of heterodimeric polypeptides where a single α chain polypeptidenoncovalently associates with a single β chain. There are now about 14distinct a chain polypeptides and at least about 8 different β chainpolypeptides which constitute the integrin family of cell adhesionreceptors. In general, different binding specificities and tissuedistributions are derived from unique combinations of the α and β chainpolypeptides or integrin subunits. The family to which a particularintegrin is associated with is usually characterized by the β subunit.However, the ligand binding activity of the integrin is largelyinfluenced by the α subunit. For example, vitronectin binding integrinscontain the α_(v) integrin subunit.

[0004] It is now known that the vitronectin binding integrins consist ofat least three different α_(v) containing integrins. These α_(v)containing integrins include α_(v)β₃, α_(v)β₁ and α_(v)β₅, all of whichexhibit different ligand binding specificities. For example, in additionto vitronectin, α_(v)β₃ binds to a large variety of extracellular matrixproteins including fibronectin, fibrinogen, laminin, thrombospondin, vonWillebrand factor, collagen, osteopontin and bone sialoprotein I. Theintegrin α_(v)β₁ binds to fibronectin, osteopontin and vitronectinwhereas α_(v)β₅ is known to bind to vitronectin and osteopontin.

[0005] As cell adhesion receptors, integrins are involved in a varietyof physiological processes including, for example, cell attachment, cellmigration and cell proliferation. Different integrins play differentroles in each of these biological processes and the inappropriateregulation of their function or activity can lead to variouspathological conditions. For example, inappropriate endothelial cellproliferation during neovascularization of a tumor has been found to bemediated by cells expressing vitronectin binding integrins. In thisregard, the inhibition of the vitronectin-binding integrin α_(v)β₃ alsoinhibits this process of tumor neovascularization. By this samecriteria, α_(v)β₃ has also been shown to mediate the abnormal cellproliferation associated with restenosis and granulation tissuedevelopment in cutaneous wounds, for example. Additional diseases orpathological states mediated or influenced by α_(v)β₃ include, forexample, metastasis, osteoporosis, age-related macular degeneration anddiabetic retinopathy, and inflammatory diseases such as rheumatoidarthritis and psoriasis. Thus, agents which can specifically inhibitvitronectin-binding integrins would be valuable for the therapeutictreatment of diseases.

[0006] Many integrins mediate their cell adhesive functions byrecognizing the tripeptide sequence Arg-Gly-Asp (RGD) found within alarge number of extracellular matrix proteins. A variety of approacheshave attempted to model agents after this sequence to target aparticular integrin-mediated pathology. Such approaches include, forexample, the use of RGD-containing peptides and peptide analogues whichrely on specificity to be conferred by the sequences flanking the RGDcore tripeptide sequence. Although there has been some limited success,most RGD-based inhibitors have been shown to be, at most, selective forthe targeted integrin and therefore exhibit some cross-reactivity toother non-targeted integrins. Such cross-reactive inhibitors thereforelack the specificity required for use as an efficacious therapeutic.This is particularly true for previously identified inhibitors of theintegrin α_(v)β₃.

[0007] Monoclonal antibodies on the other hand exhibit the specificityrequired to be used as an effective therapeutic. Antibodies also havethe advantage in that they can be routinely generated againstessentially any desired antigen. Moreover, with the development ofcombinatorial libraries, antibodies can now be produced faster and moreefficiently than by previously used methods within the art. The use ofcombinatorial methodology also allows for the selection of the desiredantibody along with the simultaneous isolation of the encoding heavy andlight chain nucleic acids. Thus, further modification can be performedto the combinatorial antibody without the incorporation of an additionalcloning step.

[0008] Regardless of the potential advantages associated with the use ofmonoclonal antibodies as therapeutics, these molecules nevertheless havethe drawback in that they are almost exclusively derived from non-humanmammalian organisms. Therefore, their use as therapeutics is limited bythe fact that they will normally elicit a host immune response. Methodsfor substituting the antigen binding site or complementarity determiningregions (CDRs) of the non-human antibody into a human framework havebeen described. Such methods vary in terms of which amino acid residuesshould be substituted as the CDR as well as which framework residuesshould be changed to maintain binding specificity. In this regard, it isunderstood that proper orientation of the β sheet architecture, correctpacking of the heavy and light chain interface and appropriateconformation of the CDRs are all important for preserving antigenspecificity and affinity within the grafted antibody. However, all ofthese methods require knowledge of the nucleotide and amino acidsequence of the non-human antibody and the availability of anappropriately modeled human framework.

[0009] Thus, there exists a need for the availability of nucleic acidsencoding integrin inhibitory antibodies which can be used as compatibletherapeutics in humans. For α_(v)β₃-mediated diseases, the presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0010] The invention provides a Vitaxin antibody and a LM609 graftedantibody exhibiting selective binding affinity to α_(v)β₃. The Vitaxinantibody consists of at least one Vitaxin heavy chain polypeptide and atleast one Vitaxin light chain polypeptide or functional fragmentsthereof. Also provided are the Vitaxin heavy and light chainpolypeptides and functional fragments. The LM609 grafted antibodyconsists of at least one LM609 CDR grafted heavy chain polypeptide andat least one LM609 CDR grafted light chain polypeptide or functionalfragment thereof. The invention additionally provides a high affinityLM609 grafted antibody comprising one or more CDRs having at least oneamino acid substitution, where the α_(v)β₃ binding activity of the highaffinity LM609 grafted antibody is enhanced. Nucleic acids encodingVitaxin and LM609 grafted heavy and light chains as well as nucleicacids encoding the parental non-human antibody LM609 are additionallyprovided. Functional fragments of such encoding nucleic acids aresimilarly provided. The invention also provides a method of inhibiting afunction of α_(v)β₃. The method consists of contacting α_(v)β₃ withVitaxin or a LM609 grafted antibody or functional fragments thereofunder conditions which allow binding to α_(v)β₃. Finally, the inventionprovides for a method of treating an α_(v)β₃-mediated disease. Themethod consists of administering an effective amount of Vitaxin or aLM609 grafted antibody or functional fragment thereof under conditionswhich allow binding to α_(v)β₃.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the nucleotide and deduced amino acid sequence of thevariable region of the antibody Vitaxin. FIG. 1A shows the nucleotideand deduced amino acid sequences for the Vitaxin heavy chain variableregion (Gln1-Ser117; SEQ ID NOS:1 and 2, respectively) while FIG. 1Bshows the nucleotide and deduced amino acid sequences for the Vitaxinlight chain variable region (Glu1-Lys107; SEQ ID NOS:3 and 4,respectively).

[0012]FIG. 2 shows the nucleotide and deduced amino acid sequence of thevariable region of the monoclonal antibody LM609. FIG. 2A shows thenucleotide and deduced amino acid sequence of the LM609 heavy chainvariable region (SEQ ID NOS:5 and 6, respectively). The variable regionextends from amino acid Glu1 to Ala117. FIG. 2B shows the nucleotide anddeduced amino acid sequence of the LM609 light chain variable region(SEQ ID NOS:7 and 8, respectively). The variable region of the lightchain extends from amino acid Asp1 to Lys107.

[0013]FIG. 3 shows the competitive inhibition of LM609 IgG binding tothe integrin avb3 with recombinant LM609 Fab. Soluble recombinant murineLM609 Fab fragments were prepared from periplasmic fractions of M13bacteriophage clones muLM609M13 12 and muLM609M13 29. The periplasmsamples were serially diluted, mixed with either 1 ng/ml, 5 ng/ml, or 50ng/ml of LM609 IgG and then incubated in 96 well plates coated withpurified α_(v)β₃. Plates were washed and bound LM609 IgG detected withgoat anti-murine Fc specific antibody conjugated to alkalinephosphatase. Fab produced by clone muLM609M13 12 inhibits both 1 ng/mland 5 ng/ml LM609 IgG binding at all concentrations of Fab greater than1:27 dilution.

[0014]FIG. 4 shows the characterization of Vitaxin binding specificity.FIG. 4A shows specific binding of Vitaxin to the integrin α_(v)β₃compared to integrins α_(IIb)β₃ and α_(v)β₅. FIG. 4B shows thecompetitive inhibition of LM609 binding toα_(v)β₃ by Vitaxin. FIG. 4Cshows the competitive inhibition of fibrinogen binding to α_(v)β₃ byVitaxin.

[0015]FIG. 5 shows the inhibition of α_(v)β₃-mediated cell attachment(5A) and migration (5B) by Vitaxin.

[0016]FIG. 6 shows the reduction in tumor growth due to Vitaxin mediatedinhibition of neovascularization. FIG. 6A shows the inhibition of theα_(v)β₃-negative Fg and HEp-3 human tumor fragments grown on chickchorioallantoic membranes (CAMs) following Vitaxin treatment. FIG. 6Bshows the growth inhibition of Vx2 carcinomas implanted subcutaneouslyin rabbits at two different Vitaxin doses administered 1 day postimplantation. FIG. 6C similarly shows Vx2 tumor growth inhibition as inFIG. 6B, except that four different Vitaxin doses were administeredbeginning at 7 days post implantation.

[0017]FIG. 7 shows the nucleotide and deduced amino acid sequence of thelight chain variable region of the LM609 grafted antibody fragment(Glu1-Lys107; SEQ ID NOS:31 and 32, respectively). Position 49 of thelight chain variable region can at least be either Arg or Met. Thenucleotide and deduced amino acid sequence of the heavy chain variableregion of the LM609 grafted antibody fragment is shown in FIG. 1A (SEQID NOS:1 and 2, respectively).

[0018]FIG. 8 shows the titration of LM609 grafted antibody variants andLM609 grafted Fab on immobilized α_(v)β₃. Bacterial cell lysatescontaining LM609 grafted antibody (closed circles), LM609 graftedantibody variants with improved affinity isolated from the primarylibraries (S102, closed squares; Y100, open squares; and Y101, opentriangles) or from combinatorial libraries (closed triangles), or anirrelevant Fab (open circles) were titrated on immobilized α_(v)β₃.

[0019]FIG. 9 shows the construction of combinatorial libraries ofenhanced LM609 grafted antibody variants containing multiple amino acidsubstitutions.

[0020]FIG. 10 shows the inhibition of fibrinogen binding to α_(v)β₃ byLM609 grafted antibody variants. FIG. 10A shows inhibition of fibrinogenbinding to immobilized α_(v)β₃. FIG. 1B shows correlation of affinity ofantibody variants with inhibition of fibrinogen binding.

[0021]FIG. 11 shows the inhibition of M21 human melanoma cell adhesionto fibrinogen by LM609 grafted antibody variants. Cell binding to 10μg/ml fibrinogen-coated substrate was assessed in the presence ofvarious concentrations of LM609 grafted Fab (closed triangles) or theenhanced LM609 grafted Fabs S102 (open circles), G102 (closed circles),or C37 (open triangles).

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention is directed to nucleic acids encoding themonoclonal antibody (MAb) LM609. This antibody specifically recognizesthe integrin α_(v)β₃ and inhibits its functional activity. The inventionis also directed to nucleic acids encoding and to polypeptidescomprising non-murine grafted forms of LM609. These grafted antibodiesretain the binding specificity and inhibitory activity of the parentmurine antibody LM609. The invention is additionally directed tooptimized forms of LM609 grafted antibodies that exhibit increasedbinding affinity and specificity compared to the non-mouse parentalforms of the LM609 grafted antibody.

[0023] In one embodiment, the hybridoma expressing LM609 was used as asource to generate and clone cDNAs encoding LM609. The heavy and lightchain encoding cDNAs were sequenced and their CDR regions weresubstituted into a human antibody framework to generate the non-murineform of the antibody. The substitution or grafting of the CDRs wasperformed by codon-based mutagenesis to generate a combinatorialantibody Fab library consisting of members that presented alternativeresidues at certain positions. Screening of the library resulted in theisolation of Vitaxin. As a grafted antibody containing human frameworksequences, it is unlikely that Vitaxin will elicit a host immuneresponse and can therefore be advantageously used for the treatment ofα_(v)β₃-mediated diseases.

[0024] As used herein, the term “monoclonal antibody LM609” or “LM609”is intended to mean the murine monoclonal antibody specific for theintegrin α_(v)β₃ which is described by Cheresh, D. A. Proc. Natl. Acad.Sci. USA 84:6471-6475 (1987) and by Cheresh and Spiro J. Biol. Chem.262:17703-17711 (1987). LM609 was produced against and is reactive withthe M21 cell adhesion receptor now known as the integrin α_(v)β₃. LM609inhibits the attachment of M21 cells to α_(v)β₃ ligands such asvitronectin, fibrinogen and von Willebrand factor (Cheresh and Spiro,supra) and is also an inhibitor of α_(v)β₃-mediated pathologies such astumor induced angiogenesis (Brooks et al. Cell 79:1157-1164 (1994),granulation tissue development in cutaneous wound (Clark et al., Am. J.Pathology, 148:1407-1421 (1996)) and smooth muscle cell migration suchas that occurring during restenosis (Choi et al., J. Vascular Surg.,19:125-134 (1994); Jones et al., Proc. Natl. Acad. Sci. 93:2482-2487(1996)).

[0025] As used herein, the term “Vitaxin” is intended to refer to anon-mouse antibody or functional fragment thereof having substantiallythe same heavy and light chain CDR amino acid sequences as found inLM609. The term “Vitaxin” when used in reference to heavy or light chainpolypeptides is intended to refer to a non-mouse heavy or light chain orfunctional fragment thereof having substantially the same heavy or lightchain CDR amino acid sequences as found in the heavy or light chain ofLM609, respectively. When used in reference to a functional fragment,not all LM609 CDRs need to be represented. Rather, only those CDRs thatwould normally be present in the antibody portion that corresponds tothe functional fragment are intended to be referenced as the LM609 CDRamino acid sequences in the Vitaxin functional fragment. Similarly, theuse of the term “Vitaxin” in reference to an encoding nucleic acid isintended to refer to a nucleic acid encoding a non-mouse antibody orfunctional fragment having substantially the same nucleotide sequence asthe heavy and light chain CDR nucleotide sequences and encodingsubstantially the same CDR amino acid sequences as found in LM609.

[0026] As used herein, the term “LM609 grafted antibody” is intended torefer to a non-mouse antibody or functional fragment thereof havingsubstantially the same heavy and light chain CDR amino acid sequences asfound in LM609 and absent of the substitution of LM609 amino acidresidues outside of the CDRs as defined by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983). The term “LM609 grafted antibody” or “LM609 grafted”when used in reference to heavy or light chain polypeptides is intendedto refer to a non-mouse heavy or light chain or functional fragmentthereof having substantially the same heavy or light chain CDR aminoacid sequences as found in the heavy or light chain of LM609,respectively, and also absent of the substitution of LM609 residuesoutside of the CDRs as defined by Kabat et al., supra. When used inreference to a functional fragment, not all LM609 CDRs need to berepresented. Rather, only those CDRs that would normally be present inthe antibody portion that corresponds to the functional fragment areintended to be referenced as the LM609 CDR amino acid sequences in theLM609 grafted functional fragment. Similarly, the term “LM609 graftedantibody” or “LM609 grafted” used in reference to an encoding nucleicacid is intended to refer to a nucleic acid encoding a non-mouseantibody or functional fragment being absent of the substitution ofLM609 amino acids outside of the CDRs as defined by Kabat et al., supraand having substantially the same nucleotide sequence as the heavy andlight chain CDR nucleotide sequences and encoding substantially the sameCDR amino acid sequences as found in LM609 and as defined by Kabat etal., supra.

[0027] The term “grafted antibody” or “grafted” when used in referenceto heavy or light chain polypeptides or functional fragments thereof isintended to refer to a heavy or light chain or functional fragmentthereof having substantially the same heavy or light chain CDR of adonor antibody, respectively, and also absent of the substitution ofdonor amino acid residues outside of the CDRs as defined by Kabat etal., supra. When used in reference to a functional fragment, not alldonor CDRs need to be represented. Rather, only those CDRs that wouldnormally be present in the antibody portion that corresponds to thefunctional fragment are intended to be referenced as the donor CDR aminoacid sequences in the functional fragment. Similarly, the term “graftedantibody” or “grafted” when used in reference to an encoding nucleicacid is intended to refer to a nucleic acid encoding an antibody orfunctional fragment, being absent of the substitution of donor aminoacids outside of the CDRs as defined by Kabat et al., supra and havingsubstantially the same nucleotide sequence as the heavy and light chainCDR nucleotide sequences and encoding substantially the same CDR aminoacid sequences as found in the donor antibody and as defined by Kabat etal., supra.

[0028] The meaning of the above terms are intended to include minorvariations and modifications of the antibody so long as its functionremains uncompromised. Functional fragments such as Fab, F(ab)₂, Fv,single chain Fv (scFv) and the like are similarly included within thedefinition of the terms LM609 and Vitaxin. Such functional fragments arewell known to those skilled in the art. Accordingly, the use of theseterms in describing functional fragments of LM609 or the Vitaxinantibody are intended to correspond to the definitions well known tothose skilled in the art. Such terms are described in, for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1989); Molec. Biology and Biotechnology: AComprehensive Desk Reference (Myers, R. A. (ed.), New York: VCHPublisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993);Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York,N.Y. (1990).

[0029] As with the above terms used for describing functional fragmentsof LM609, Vitaxin and a LM609 grafted antibody, the use of terms whichreference other LM609, Vitaxin or LM609 grafted antibody domains,functional fragments, regions, nucleotide and amino acid sequences andpolypeptides or peptides, is similarly intended to fall within the scopeof the meaning of each term as it is known and used within the art. Suchterms include, for example, “heavy chain polypeptide” or “heavy chain”,“light chain polypeptide” or “light chain”, “heavy chain variableregion” (V_(H)) and “light chain variable region” (V_(L)) as well as theterm “complementarity determining region” (CDR).

[0030] In the case where there are two or more definitions of a termwhich is used and/or accepted within the art, the definition of the termas used herein is intended to include all such meanings unlessexplicitly stated to the contrary. A specific example is the use of theterm “CDR” to describe the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.This particular region has been described by Kabat et al., supra, and byChothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum etal., J. Mol. Biol. 262:732-745 (1996) where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof LM609, Vitaxin, LM609 grafted antibodies or variants thereof isintended to be within the scope of the term as defined and used herein.The amino acid residues which encompass the CDRs as defined by each ofthe above cited references are set forth below in Table 1 as acomparison. TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H)CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102 96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-5246-55 V_(L) CDR3 89-97 91-96 89-96

[0031] As used herein, the term “substantially” or “substantially thesame” when used in reference to a nucleotide or amino acid sequence isintended to mean that the nucleotide or amino acid sequence shows aconsiderable degree, amount or extent of sequence identity when comparedto a reference sequence. Such considerable degree, amount or extent ofsequence identity is further considered to be significant and meaningfuland therefore exhibit characteristics which are definitivelyrecognizable or known. Thus, a nucleotide sequence which issubstantially the same nucleotide sequence as a heavy or light chain ofLM609, Vitaxin, or a LM609 grafted antibody including fragments thereof,refers to a sequence which exhibits characteristics that aredefinitively known or recognizable as encoding or as being the aminoacid sequence of LM609, Vitaxin or a LM609 grafted antibody. Minormodifications thereof are included so long as they are recognizable as aLM609, Vitaxin or a LM609 grafted antibody sequence. Similarly, an aminoacid sequence which is substantially the same amino acid sequence as aheavy or light chain of Vitaxin, a LM609 grafted antibody or functionalfragment thereof, refers to a sequence which exhibits characteristicsthat are definitively known or recognizable as representing the aminoacid sequence of Vitaxin or a LM609 grafted antibody and minormodifications thereof.

[0032] When determining whether a nucleotide or amino acid sequence issubstantially the same as Vitaxin or a LM609 grafted antibody,consideration is given to the number of changes relative to the Vitaxinor LM609 grafted antibody together with whether the function ismaintained. For example, a single amino acid change in a 3 amino acidCDR or several changes in a 16 amino acid CDR are considered to besubstantially the same if α_(v)β₃ binding function is maintained. Thus,a nucleotide or amino acid sequence is substantially the same if itexhibits characteristics that are definitively known or recognizable asrepresenting the nucleotide or amino acid sequence of Vitaxin or a LM609grafted antibody and minor modifications thereof as long as Vitaxin orLM609 grafted antibody function is maintained.

[0033] As used herein, the term “fragment” when used in reference to anucleic acid encoding LM609, Vitaxin or a LM609 grafted antibody isintended to mean a nucleic acid having substantially the same sequenceas a portion of a nucleic acid encoding LM609, Vitaxin or a LM609grafted antibody. The nucleic acid fragment is sufficient in length andsequence to selectively hybridize to an LM609, a Vitaxin or a LM609grafted antibody encoding nucleic acid or a nucleotide sequence that iscomplementary to an LM609, Vitaxin or LM609 grafted antibody encodingnucleic acid. Therefore, fragment is intended to include primers forsequencing and polymerase chain reaction (PCR) as well as probes fornucleic acid blot or solution hybridization. The meaning of the term isalso intended to include regions of nucleotide sequences that do notdirectly encode LM609 polypeptides such as the introns, and theuntranslated region sequences of the LM609 encoding gene.

[0034] As used herein, the term “functional fragment” when used inreference to Vitaxin, to a LM609 grafted antibody or to heavy or lightchain polypeptides thereof is intended to refer to a portion of Vitaxinor a LM609 grafted antibody including heavy or light chain polypeptideswhich still retains some or all or the α_(v)β₃ binding activity, α_(v)β₃binding specificity and/or integrin α_(v)β₃-inhibitory activity. Suchfunctional fragments can include, for example, antibody functionalfragments such as Fab, F(ab)₂, Fv, single chain Fv (scFv). Otherfunctional fragments can include, for example, heavy or light chainpolypeptides, variable region polypeptides or CDR polypeptides orportions thereof so long as such functional fragments retain bindingactivity, specificity or inhibitory activity. The term is also intendedto include polypeptides encompassing, for example, modified forms ofnaturally occurring amino acids such as D-stereoisomers, non-naturallyoccurring amino acids, amino acid analogues and mimetics so long as suchpolypeptides retain functional activity as defined above.

[0035] As used herein, the term “enhanced” when used in reference toVitaxin, a LM609 grafted antibody or a functional fragment thereof isintended to mean that a functional characteristic of the antibody hasbeen altered or augmented compared to a reference antibody so that theantibody exhibits a desirable property or activity. An antibodyexhibiting enhanced activity can exhibit, for example, higher affinityor lower affinity binding, or increased or decreased association ordissociation rates compared to a reference antibody. An antibodyexhibiting enhanced activity can also exhibit increased stability suchas increased half-life in a particular organism. For example, if higheraffinity binding is desired, mutations can be introduced into frameworkor CDR amino acid residues and the resulting antibody variants screenedfor higher affinity binding to α_(v)β₃ relative to a reference antibodysuch as the LM609 grafted parent antibody.

[0036] The invention provides a nucleic acid encoding a heavy chainpolypeptide for Vitaxin or a functional fragment thereof. Also providedis a nucleic acid encoding a light chain polypeptide for Vitaxin or afunctional fragment thereof. The nucleic acids consist of substantiallythe same heavy or light chain variable region nucleotide sequences asthose shown in FIGS. 1A and 1B (SEQ ID NOS:1 and 3, respectively) or afragment thereof.

[0037] Vitaxin, including functional fragments thereof, is a non-mouseantibody which exhibits substantially the same binding activity, bindingspecificity and inhibitory activity as LM609. The Vitaxin Fv Fragmentwas produced by functionally replacing CDRs within human heavy and lightchain variable region polypeptides with the CDRs derived from LM609.Functional replacement of the CDRs was performed by recombinant methodsknown to those skilled in the art. Such methods are commonly referred toas CDR grafting and are the subject matter of U.S. Pat. No. 5,225,539.Such methods can also be found described in “Protein Engineering ofAntibody Molecules for Prophylactic and Therapeutic Applications inMan,” Clark, M. (ed.), Nottingham, England: Academic Titles (1993).

[0038] Briefly, LM609 nucleic acid fragments having substantially thesame nucleotide and encoding substantially the same amino acid sequenceof each of the heavy and light chain CDRs were synthesized andsubstituted into each of the respective human chain encoding nucleicacids. To maintain functionality of the newly derived Vitaxin antibody,modifications were performed within the non-CDR framework region. Theseindividual changes were made by generating a population of CDR graftedheavy and light chain variable regions wherein all possible changesoutside of the CDRs were represented and then selecting the appropriateantibody by screening the population for binding activity. This screenresulted in the selection of the Vitaxin antibody described herein.

[0039] The nucleotide sequences of the Vitaxin heavy and light chainvariable regions are shown in FIGS. 1A and 1B, respectively. Thesesequences correspond substantially to those that encode the heavy andlight chain variable region polypeptides of Vitaxin. These Vitaxinnucleic acids are intended to include both the sense and anti-sensestrands of the Vitaxin encoding sequences. Single- and double-strandednucleic acids are similarly included as well as non-coding portions ofthe nucleic acid such as introns, 5′- and 3′-untranslated regions andregulatory sequences of the gene for example.

[0040] As shown in FIG. 1A, the Vitaxin heavy chain variable regionpolypeptide is encoded by a nucleic acid of about 351 nucleotides inlength which begins at the amino terminal Gln1 residue of the variableregion through to Ser117. This Vitaxin heavy chain variable regionencoding nucleic acid is joined to a human IgG1 constant region to yielda coding region of 1431 nucleotides which encodes a heavy chainpolypeptide of 477 total amino acids. Shown in FIG. 1B is the Vitaxinlight chain variable region polypeptide which is encoded by a nucleicacid of about 321 nucleotides in length beginning at the amino terminalGlu1 residue of the variable region through to Lys107. This Vitaxinlight chain variable region nucleic acid is joined to a human kappaconstruct region to yield a coding region of 642 nucleotides which codefor a light chain polypeptide of 214 total amino acids.

[0041] Minor modification of these nucleotide sequences are intended tobe included as heavy and light chain Vitaxin encoding nucleic acids andtheir functional fragments. Such minor modifications include, forexample, those which do not change the encoded amino acid sequence dueto the degeneracy of the genetic code as well as those which result inonly a conservative substitution of the encoded amino acid sequence.Conservative substitutions of encoded amino acids include, for example,amino acids which belong within the following groups: (1) non-polaramino acids (Gly, Ala, Val, Leu, and Ile); (2) polar neutral amino acids(Cys, Met, Ser, Thr, Asn, and Gln); (3) polar acidic amino acids (Aspand Glu); (4) polar basic amino acids (Lys, Arg and His); and (5)aromatic amino acids (Phe, Trp, Tyr, and His). Other minor modificationsare included within the nucleic acids encoding Vitaxin heavy and lightchain polypeptides so long as the nucleic acid or encoded polypeptidesretain some or all of their function as described herein.

[0042] Thus, the invention also provides a nucleic acid encoding aVitaxin heavy chain or functional fragment thereof wherein the nucleicacid encodes substantially the same heavy chain variable region aminoacid sequence of Vitaxin as that shown in FIG. 1A (SEQ ID NO:2) or afragment thereof. Similarly, the invention also provides a nucleic acidencoding a Vitaxin light chain or functional fragment thereof whereinthe nucleic acid encodes substantially the same light chain variableregion amino acid sequence of Vitaxin as that shown in FIG. 1B (SEQ IDNO:4) or a fragment thereof.

[0043] In addition to conservative substitutions of amino acids, minormodifications of the Vitaxin encoding nucleotide sequences which allowfor the functional replacement of amino acids are also intended to beincluded within the definition of the term. The substitution offunctionally equivalent amino acids encoded by the Vitaxin nucleotidesequences is routine and can be accomplished by methods known to thoseskilled in the art. Briefly, the substitution of functionally equivalentamino acids can be made by identifying the amino acids which are desiredto be changed, incorporating the changes into the encoding nucleic acidand then determining the function of the recombinantly expressed andmodified Vitaxin polypeptide or polypeptides. Rapid methods for makingand screening multiple simultaneous changes are well known within theart and can be used to produce a library of encoding nucleic acids whichcontain all possible or all desired changes and then expressing andscreening the library for Vitaxin polypeptides which retain function.Such methods include, for example, codon based mutagenesis, randomoligonucleotide synthesis and partially degenerate oligonucleotidesynthesis.

[0044] Codon based mutagenesis is the subject matter of U.S. Pat. Nos.5,264,563 and 5,523,388 and is advantageous for the above proceduressince it allows for the production of essentially any and all desiredfrequencies of encoded amino acid residues at any and all particularcodon positions within an oligonucleotide. Such desired frequenciesinclude, for example, the truly random incorporation of all twenty aminoacids or a specified subset thereof as well as the incorporation of apredetermined bias of one or more particular amino acids so as toincorporate a higher or lower frequency of the biased residues comparedto other incorporated amino acid residues. Random oligonucleotidesynthesis and partially degenerate oligonucleotide synthesis cansimilarly be used for producing and screening for functionallyequivalent amino acid changes. However, due to the degeneracy of thegenetic code, such methods will incorporate redundancies at a desiredamino acid position. Random oligonucleotide synthesis is the coupling ofall four nucleotides at each nucleotide position within a codon whereaspartially degenerate oligonucleotide synthesis is the coupling of equalportions of all four nucleotides at the first two nucleotide positions,for example, and equal portions of two nucleotides at the thirdposition. Both of these latter synthesis methods can be found describedin, for example, Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382,(1990) and Devlin et al., Science 249:404-406, (1990).

[0045] Identification of amino acids to be changed can be accomplishedby those skilled in the art using current information availableregarding the structure and function of antibodies as well as availableand current information encompassing methods for CDR graftingprocedures. For example, CDRs can be identified within the donorantibody by any or all of the criteria specified in Kabat et al., supra,Chothia et al., supra, and/or MacCallum et al., supra, and any or allnon-identical amino acid residues falling outside of these CDR sequencescan be changed to functionally equivalent amino acids. Using the abovedescribed methods known within the art, any or all of the non-identicalamino acids can be changed either alone or in combination with aminoacids at different positions to incorporate the desired number of aminoacid substitutions at each of the desired positions. The Vitaxinpolypeptides containing the desired substituted amino acids are thenproduced and screened for retention or augmentation of function comparedto the unsubstituted Vitaxin polypeptides. Production of the substitutedVitaxin polypeptides can be accomplished by, for example, recombinantexpression using methods known to those skilled in the art. ThoseVitaxin polypeptides which exhibit retention or augmentation of functioncompared to unsubstituted Vitaxin are considered to contain minormodifications of the encoding nucleotide sequence which result in thefunctional replacement of one or more amino acids.

[0046] The functional replacement of amino acids is beneficial whenproducing grafted antibodies having human framework sequences since itallows for the rapid identification of equivalent amino acid residueswithout the need for structural information or the laborious proceduresnecessary to assess and identify which amino acid residues should beconsidered for substitution in order to successfully transfer bindingfunction from the donor. Moreover, it eliminates the actual step-wiseprocedures to change and test the amino acids identified forsubstitution. Essentially, using the functional replacement approachdescribed above, all non-identical amino acid residues between the donorand the human framework can be identified and substituted with any orall other possible amino acid residues at each non-identical position toproduce a population of substituted polypeptides containing all possibleor all desired permutations and combinations. The population ofsubstituted polypeptides can then be screened for those substitutedpolypeptides which retain function. Using the codon based mutagenesisprocedures described above, the generation of a library of substitutedamino acid residues and the screening of functionally replaced residueshas been used for the rapid production of grafted therapeutic antibodiesas well as for the rapid alteration of antibody affinity. Suchprocedures are exemplified in, for example, Rosok et al., J. Biol. Chem.271:22611-22618 (1996) and in Glaser et al., J. Immunol. 149:3903-3913(1992), respectively.

[0047] In addition to framework residues, amino acids in one or moreCDRs can be functionally replaced to allow identification of a modifiedLM609 grafted antibody having enhanced activity. Using the methodsdescribed above for framework residues, amino acid substitutions cansimilarly be introduced into one or more CDRs in an LM609 graftedantibody. The modified LM609 grafted antibody can be tested for bindingactivity to determine whether α_(v)β₃ binding activity is maintained.The modified LM609 grafted antibody can be further tested to determineif activity has been enhanced. Functional replacement of amino acidresidues in one or more CDRs therefore allows the identification of anenhanced LM609 grafted antibody having a desirable property such asenhanced activity.

[0048] To generate modified LM609 grafted antibodies and select thosewith enhanced activity, several approaches can be employed in theselection of the number of residues within a CDR to mutate as well asthe number of CDRs within a LM609 grafted antibody to modify. The choiceof selection criteria for mutagenesis of CDRs will depend on the needand desired application of the enhanced antibody. For example, one or afew amino acid positions within a single CDR can be modified to containselected amino acids at that position. Alternatively, the targeted aminoacid positions can be modified to contain all possible amino acids atthat position. The resultant population of modified antibodies can thenbe screened for enhanced activity.

[0049] The construction of modified LM609 grafted antibody populationscan also be made where all amino acids positions within a CDR have beenmutated to contain all possible amino acids and where amino acidpositions within multiple CDRs have been modified to contain variantamino acid residues. In this way, populations can be constructed whichrange from 2 to >10⁷ unique members. The larger the population, the moreefficient will be the selection of an enhanced LM609 grafted antibodysince there will be a larger number of different antibodies within thepopulation. However, a small population of modified LM609 graftedantibodies can be made and successfully used for the selection ofenhanced LM609 grafted antibodies. The size of the population ofmodified LM609 grafted antibodies will depend on the need of aparticular application and can be determined by one skilled in the art.

[0050] The generation of modified LM609 grafted antibodies can beachieved by introducing amino acid substitutions into one or more CDRsof an LM609 grafted antibody. For example, single amino acidsubstitutions can be systematically introduced into a CDR by changing agiven amino acid in the CDR to any or all amino acids. Amino acidsubstitutions can also be introduced into all amino acid positions inone or more of the CDRs or in all of the CDRs, generating a populationof modified LM609 grafted antibody variants. This population of modifiedLM609 grafted antibody variants having single amino acid substitutionscan be screened to identify those variants that maintain α_(v)β₃ bindingactivity. The variants having α_(v)β₃ binding activity can be furthercharacterized to identify those variants having enhanced activity. Sucha systematic approach to introducing single amino acid substitutions andgenerating a population of LM609 grafted antibody variants to screen forenhanced LM609 grafted antibodies having high affinity binding toα_(v)β₃ is described in Example VI.

[0051] In addition to generating a population of modified LM609 graftedantibody variants, a particular CDR or a particular amino acid in a CDRcan be selected to introduce one or more amino acid substitutions. Forexample, sequence homology or a structural model can be used to identifyparticular amino acid positions to introduce amino acid substitutions.In this example, only one or a few modified LM609 grafted antibodyvariants are generated and screened for binding activity to α_(v)β₃. Oneof skill in the art will know or can determine whether it is desirableto generate a large population of modified LM609 grafted antibodyvariants or to generate a limited number of modified LM609 graftedantibody variants to screen and identify an enhanced LM609 graftedantibody having enhanced activity.

[0052] In addition to identifying enhanced LM609 grafted antibodies bygenerating a population of modified LM609 grafted antibodies havingsingle amino acid substitutions in a CDR and screening for enhancedactivity, enhanced LM609 grafted antibody variants can also be generatedby combining two or more mutations, each known to independently resultin enhanced activity, into a single antibody. When there are more thantwo mutations, an efficient way to identify combinations of mutationswhich further augment activity is to construct all possible combinationsand permutations and then select for those with enhanced activity. Forexample, two single mutations in one or more CDRs can be combined togenerate a new modified LM609 grafted antibody having two CDR mutationsand screened to determine if the α_(v)β₃ binding activity is increasedover that of the single mutants. Similarly, three mutations can becombined and the resulting modified LM609 grafted antibody screened forenhanced binding activity. Using such an approach of combining CDRmutations, a new population of modified LM609 grafted antibody variantscan be generated by incorporating all combinations of the single CDRmutations resulting in enhanced activity into new modified LM609 graftedantibody variants and screening to obtain an optimized enhanced LM609grafted antibody.

[0053] An iterative, step-wise approach to identifying an enhanced LM609grafted antibody is advantageous in that it allows the identification ofan antibody having optimal binding activity without the need to generateand screen a large number of modified LM609 grafted antibody variants.For example, using the approach described in Examples VI and VII inwhich single mutants were identified and combined into a new populationof LM609 grafted antibody variants, enhanced LM609 grafted antibodieshaving higher affinity were identified by generating 2592 uniquevariants. In contrast, complete randomization of a single eight aminoacid residue CDR would require >10¹⁰ unique variants. Therefore, such aniterative approach allows identification of enhanced LM609 graftedantibodies having enhanced activity such as high affinity binding bygenerating a relatively small number of unique modified LM609 graftedantibody variants and screening and identifying those enhanced LM609grafted antibody variants exhibiting high affinity binding.

[0054] An iterative, step-wise approach to identifying enhanced LM609variants can also be performed using additional steps. Instead ofgenerating all combinations of single amino acid mutations, the singleamino acid mutations can be combined in pairs to generate allcombinations of double mutants and screened for activity. Those doublemutants having enhanced activity can be combined with any or all singlemutants to generate triple mutants that are screened for enhancedbinding activity. Each iterative round of generating modified LM609grafted antibody variants can incorporate additional single mutations,and the resulting modified LM609 grafted antibodies can be screened forenhanced activity. The step-wise generation of LM609 grafted antibodyvariants can thus be used to identify an optimized LM609 graftedantibody. Additionally, such an iterative approach also allows for theidentification of numerous enhanced antibodies which exhibit a range ofdifferent, enhanced binding activities.

[0055] An optimized LM609 grafted antibody can also be referred to as anLM609-like grafted antibody or an α_(v)β₃-specific grafted antibody andis recognizable because the antibody or functional fragments thereofretains the functional characteristics of LM609. For example, enhancedLM609 grafted antibody variants, which have a single amino acidsubstitution and have enhanced activity, can be identified andcorrelated with a specific amino acid substitution. These amino acidsubstitutions can be combined to generate a new modified LM609 graftedantibody that is tested for activity. Such a combination of advantageousCDR amino acid substitutions can result in an optimized LM609 graftedantibody with multiple CDRs having at least one amino acid substitutionor a single CDR having multiple amino acid substitutions, where themodified LM609 grafted antibody has enhanced activity.

[0056] Enhanced LM609 grafted antibodies, particularly those optimizedby functional replacement of amino acid residues in the CDRs, havedesirable enhanced properties such as increased affinity. For example,an optimized LM609 grafted antibody having increased affinity will havehigher affinity than the parent antibody used for introducing functionalreplacement of amino acids. Higher affinity is determined relative to areference antibody having a similar structure. For example, if theoptimized LM609 grafted antibody is an intact antibody containing twoheavy chains and two light chains, then higher affinity is determinedrelative to the intact parent LM609 grafted antibody. Similarly, if theoptimized LM609 grafted antibody is an Fab, then higher affinity isdetermined relative to the Fab of the parent LM609 grafted antibody.

[0057] Although it is not necessary to proceed through multipleoptimization steps to obtain a high affinity LM609 grafted antibody, ingeneral, the increase in affinity can correlate with the number ofmodifications within and between CDRs as well as with the number ofoptimization steps. Therefore, LM609 grafted antibodies will exhibit avariety of ranges. For example, LM609 grafted antibodies having enhancedaffinity will have up to about 2-fold higher affinity or greater,generally greater than about 2- to 5-fold higher affinity such asgreater than about 4- to 5-fold higher affinity or about 5- to 10-foldhigher affinity than the reference antibody. Particularly, a LM609grafted antibody having enhanced affinity will have greater than about10- to 50-fold higher affinity, greater than about 50-fold higheraffinity, or greater than about 100-fold higher affinity than thereference antibody.

[0058] As described above, functional replacement of CDR amino acidresidues can be used to identify LM609 grafted antibodies exhibitinghigher affinity than a parent LM609 grafted antibody. Methods discussedabove or below for introducing minor modifications into Vitaxin or LM609grafted antibody encoding nucleotide sequences can similarly be used togenerate a library of modified LM609 grafted antibody variants,including methods such as codon based mutagenesis, randomoligonucleotide synthesis and partially degenerate oligonucleotidesynthesis. For example, codon based mutagenesis has been used togenerate such a library of modified LM609 grafted antibody variantshaving single amino acid substitutions (see Example VI).

[0059] After generating a library of modified LM609 grafted antibodyvariants, the variants can be expressed and screened for bindingactivity to α_(v)β₃. Methods well known to those skilled in the artrelated to determining antibody-antigen interactions are used to screenfor modified LM609 grafted antibodies exhibiting binding activity toα_(v)β₃ (Harlow and Lane, supra). For example, an ELISA method has beenused to screen a library of modified LM609 grafted antibody variants toidentify those variants that maintained α_(v)β₃ binding activity (seeExample VI). Only those modified LM609 grafted antibodies that maintainα_(v)β₃ binding activity are considered for further characterization.

[0060] Modified LM609 grafted antibodies having α_(v)β₃ binding activitycan be further characterized to determine which modified LM609 graftedantibody has enhanced activity. The type of assay used to assessenhanced activity depends on the particular desired characteristic. Forexample, if altered binding activity is desired, then binding assaysthat allow determination of binding affinity are used. Such assaysinclude binding assays, competition binding assays and surface plasmonresonance as described in Example VI.

[0061] Introduction of single amino acid substitutions into CDRs ofLM609 grafted antibodies can be used to generate a library of modifiedLM609 grafted antibodies and screen for binding activity to α_(v)β₃.Those modified LM609 grafted antibodies exhibiting binding activity toα_(v)β₃ can then be further characterized to identify enhanced LM609grafted antibodies exhibiting enhanced activity such as higher bindingaffinity. For example, using such an approach, a number of enhancedLM609 grafted antibodies having single amino acid substitutions weregenerated using the heavy chain variable region shown in FIG. 1a (SEQ IDNO:2) and the light chain variable region shown in FIG. 7 (SEQ IDNO:32), and LM609 grafted antibodies were identified displaying 2 to13-fold improved affinity over the parent LM609 grafted antibody (seeExample VI).

[0062] Following identification of enhanced LM609 grafted antibodieshaving a single amino acid substitution, the amino acid mutations can becombined to further enhance activity. Methods discussed above forintroducing single amino acid substitutions into CDRs can similarly beapplied to combine amino acid substitutions. For example, acombinatorial library of amino acid mutations that resulted in enhancedα_(v)β₃ binding affinity was generated using degenerate oligonucleotidesand two site hybridization mutagenesis as described in Example VII.Enhanced LM609 grafted antibodies containing multiple CDR amino acidsubstitutions were generated using the heavy chain variable region shownin FIG. 1a (SEQ ID NO:2) and the light chain variable region shown inFIG. 7 (SEQ ID NO:32), and LM609 grafted antibodies were identifiedhaving 20-fold higher affinity to greater than 90-fold higher affinitythan the parent LM609 grafted antibody.

[0063] In addition to combining CDR amino acid substitutions to generatean enhanced or optimized LM609 grafted antibody, CDR amino acidsubstitutions can also be combined with framework mutations thatcontribute desirable properties to a LM609 grafted antibody. Thus,mutations in CDR or framework regions that enhance activity can becombined to further optimize LM609 grafted antibodies.

[0064] The invention further provides fragments of Vitaxin heavy andlight chain encoding nucleic acids wherein such fragments consistsubstantially of the same nucleotide or amino acid sequence as thevariable region of Vitaxin heavy or light chain polypeptides. Thevariable region of the Vitaxin heavy chain polypeptide consistsessentially of nucleotides 1-351 and of amino acid residues Gln1 toSer117 of FIG. 1A (SEQ ID NOS:1 and 2, respectively). The variableregion of the Vitaxin light chain polypeptide consists essentially ofnucleotides 1-321 and of amino acid residues Glu1 to Lys107 of FIG. 1B(SEQ ID NOS:3 and 4, respectively). The termini of such variable regionencoding nucleic acids is not critical so long as the intended purposeand function remains the same.

[0065] Fragments additional to the variable region nucleic acidfragments are provided as well. Such fragments include, for example,nucleic acids consisting substantially of the same nucleotide sequenceas a CDR of a Vitaxin heavy or light chain polypeptide. Sequencescorresponding to the Vitaxin CDRs include, for example, those regionsdefined by Kabat et al., supra, and/or those regions defined by Chothiaet al., supra, as well as those defined by MacCallum et al., supra. TheVitaxin CDR fragments for each of the above definitions correspond tothe nucleotides set forth below in Table 2. The nucleotide sequencenumbering is taken from the primary sequence shown in FIGS. 1A and 1B(SEQ ID NOS:1 and 3) and conforms to the definitions previously setforth in Table 1. TABLE 2 Vitaxin CDR Nucleotide Residues Kabat ChothiaMacCallum V_(H) CDR1  91-105 76-96  88-105 V_(H) CDR2 148-198 157-168139-177 V_(H) CDR3 295-318 298-315 289-315 V_(L) CDR1  70-102 76-96 88-108 V_(L) CDR2 148-168 148-156 136-165 V_(L) CDR3 265-291 271-288265-288

[0066] Similarly, the Vitaxin CDR fragments for each of the abovedefinitions correspond to the amino acid residues set forth below inTable 3. The amino acid residue number is taken from the primarysequence shown in FIGS. 1A and 1B (SEQ ID NOS:2 and 4) and conforms tothe definitions previously set forth in Table 1. TABLE 3 Vitaxin CDRAmino Acid Residues Kabat Chothia MacCallum V_(H) CDR1 Ser31-Ser35Gly26-Tyr32 Ser30-Ser35 V_(H) CDR2 Lys50-Gly66 Ser53-Gly56 Trp47-Tyr59V_(H) CDR3 His99-Tyr106 Asn100-Ala105 Ala97-Ala105 V_(L) CDR1Gln24-His34 Ser26-His32 Ser30-Tyr36 V_(L) CDR2 Tyr50-Ser56 Tyr50-Ser52Leu46-Ile55 V_(L) CDR3 Gln89-Thr97 Ser91-His96 Gln89-His96

[0067] Thus, the invention also provides nucleic acid fragments encodingsubstantially the same amino acid sequence as a CDR of a Vitaxin heavyor light chain polypeptide.

[0068] Nucleic acids encoding Vitaxin heavy and light chain polypeptidesand fragments thereof are useful for a variety of diagnostic andtherapeutic purposes. For example, the Vitaxin nucleic acids can be usedto produce Vitaxin antibodies and functional fragments thereof havingbinding specificity and inhibitory activity against the integrinα_(v)β₃. The antibody and functional fragments thereof can be used forthe diagnosis or therapeutic treatment of α_(v)β₃-mediated disease.Vitaxin and functional fragments thereof can be used, for example, toinhibit binding activity or other functional activities of α_(v)β₃ thatare necessary for progression of an α_(v)β₃-mediated disease. Otherfunctional activities necessary for progression of α_(v)β₃-mediateddisease include, for example, the activation of α_(v)β₃,α_(v)β₃-mediated signal transduction and the α_(v)β₃-mediated preventionof apoptosis. Advantageously, however, Vitaxin comprises non-mouseframework amino acid sequences and as such is less antigenic in regardto the induction of a host immune response. The Vitaxin nucleic acids ofthe inventions can also be used to model functional equivalents of theencoded heavy and light chain polypeptides.

[0069] Thus, the invention provides Vitaxin heavy chain and Vitaxinlight chain polypeptides or functional fragments thereof. The Vitaxinheavy chain polypeptide exhibits substantially the same amino acidsequence as that shown in FIG. 1A (SEQ ID NO:2) or functional fragmentthereof whereas the Vitaxin light chain polypeptide exhibitssubstantially the same amino acid sequence as that shown in FIG. 1B (SEQID NO:4) or functional fragment thereof. Also provided is a Vitaxinantibody or functional fragment thereof. The antibody is generated fromthe above heavy and light chain polypeptides or functional fragmentsthereof and exhibits selective binding affinity to α_(v)β₃.

[0070] The invention provides a nucleic acid encoding a heavy chainpolypeptide for a LM609 grafted antibody. Also provided is a nucleicacid encoding a light chain polypeptide for a LM609 grafted antibody.The nucleic acids consist of substantially the same heavy chain variableregion nucleotide sequence as that shown in FIG. 1A (SEQ ID NO:1) andsubstantially the same light chain variable region nucleotide sequenceas that shown in FIG. 7 (SEQ ID NO:31) or a fragment thereof.

[0071] LM609 grafted antibodies, including functional fragments thereof,are non-mouse antibodies which exhibit substantially the same bindingactivity, binding specificity and inhibitory activity as LM609. TheLM609 grafted antibody Fv fragments described herein are produced byfunctionally replacing the CDRs as defined by Kabat et al., hereinafterreferred to as “Kabat CDRs,” within human heavy and light chain variableregion polypeptides with the Kabat CDRs derived from LM609. Functionalreplacement of the Kabat CDRs is performed by the CDR grafting methodspreviously described and which is the subject matter of U.S. Pat. No.5,225,539, supra. Substitution of amino acid residues outside of theKabat CDRs can additionally be performed to maintain or augmentbeneficial binding properties so long as such amino acid substitutionsdo not correspond to a donor amino acid at that particular position.Such substitutions allow for the modulation of binding propertieswithout imparting any mouse sequence characteristics onto the antibodyoutside of the Kabat CDRs. Although the production of such antibodies isdescribed herein with reference to LM609 grafted antibodies, thesubstitution of such non-donor amino acids outside of the Kabat CDRs canbe utilized for the production of essentially any grafted antibody. Theproduction of LM609 grafted antibodies is described further below inExample V.

[0072] The nucleotide sequences of the LM609 grafted antibody heavy andlight chain variable regions are shown in FIGS. 1A and 7, respectively.These sequences correspond substantially to those that encode the heavyand light chain variable region polypeptides of a LM609 graftedantibody. These nucleic acids are intended to include both the sense andanti-sense strands of the LM609 grafted antibody encoding sequences.Single- and double-stranded nucleic acids are similarly included as wellas non-coding portions of the nucleic acid such as introns, 5′- and3′-untranslated regions and regulatory sequences of the gene forexample.

[0073] The nucleotide and amino acid residue boundaries for a LM609grafted antibody are identical to those previously described forVitaxin. For example, a LM609 grafted antibody heavy chain variableregion polypeptide is encoded by a nucleic acid of about 351 nucleotidesin length which begins at the amino terminal Gln1 residue of thevariable region through to Ser117 (FIG. 1A, SEQ ID NOS:1 and 2,respectively). The LM609 grafted antibody light chain variable regionpolypeptide is encoded by a nucleic acid of about 321 nucleotides inlength beginning at the amino terminal Glu1 residue of the variableregion through to Lys107 (FIG. 7, SEQ ID NOS:31 and 32, respectively).As with Vitaxin, minor modification of these nucleotide sequences areintended to be included as heavy and light chain variable regionencoding nucleic acids and their functional fragments.

[0074] Thus, the invention also provides a nucleic acid encoding a LM609grafted antibody heavy chain wherein the nucleic acid encodessubstantially the same heavy chain variable region amino acid sequenceas that shown in FIG. 1A (SEQ ID NO:2) or fragment thereof. Similarly,the invention also provides a nucleic acid encoding a LM609 graftedantibody light chain wherein the nucleic acid encodes substantially thesame light chain variable region amino acid sequence as that shown inFIG. 7 (SEQ ID NO:32) or fragment thereof.

[0075] In addition to conservative substitutions of amino acids, minormodifications of the LM609 grafted antibody encoding nucleotidesequences which allow for the functional replacement of amino acids arealso intended to be included within the definition of the term.Identification of amino acids to be changed can be accomplished by thoseskilled in the art using current information available regarding thestructure and function of antibodies as well as available and currentinformation encompassing methods for CDR grafting procedures. Thesubstitution of functionally equivalent amino acids encoded by the LM609grafted antibody nucleotide sequences is routine and can be accomplishedby methods known to those skilled in the art. As described previously,such methods include, for example, codon based mutagenesis, randomoligonucleotide synthesis and partially degenerate oligonucleotidesynthesis and are beneficial when producing grafted antibodies sincethey allow for the rapid identification of equivalent amino acidresidues without the need for structural information.

[0076] The invention further provides fragments of LM609 graftedantibody heavy and light chain encoding nucleic acids wherein suchfragments consist substantially of the same nucleotide or amino acidsequence as the variable region of a LM609 grafted antibody heavy orlight chain polypeptide. As with Vitaxin, the termini of such variableregion encoding nucleic acids is not critical so long as the intendedpurpose and function remains the same.

[0077] Fragments additional to the variable region nucleic acidfragments are provided as well and include, for example, nucleic acidsconsisting substantially of the same nucleotide sequence as a CDR of aLM609 grafted antibody heavy or light chain polypeptide. As withVitaxin, sequences corresponding to the LM609 grafted antibody CDRsinclude, for example, those regions defined by Kabat et al., supra,Chothia et al., supra, as well as those defined by MacCallum et al.,supra. The LM609 grafted antibody CDR regions will be similar to thosedescribed previously for Vitaxin. Moreover, such regions are well knownand can be determined by those skilled in the art given the LM609sequences and teachings provided herein. Thus, the invention alsoprovides nucleic acid fragments encoding substantially the same aminoacid sequence as a CDR of a LM609 grafted antibody heavy or light chainpolypeptide.

[0078] As with Vitaxin, nucleic acids encoding LM609 grafted antibodyheavy and light chain polypeptides and fragments thereof are useful fora variety of diagnostic and therapeutic purposes. For example, LM609grafted antibody encoding nucleic acids can be used to producerecombinant antibodies and functional fragments thereof having bindingspecificity and inhibitory activity against the integrin α_(v)β₃. Theantibody and functional fragments thereof can be used for the diagnosisor therapeutic treatment of α_(v)β₃-mediated disease. Such diseases andmethods of use for anti-α_(v)β₃ antibodies have been describedpreviously in reference to Vitaxin and are equally applicable to theLM609 grafted antibodies described herein.

[0079] Thus, the invention provides LM609 grafted antibody heavy chainand Vitaxin light chain polypeptides or functional fragments thereof.The LM609 grafted antibody heavy chain polypeptide exhibitssubstantially the same amino acid sequence as that shown in FIG. 1A (SEQID NO:2) or functional fragment thereof whereas the LM609 graftedantibody light chain polypeptide exhibits substantially the same aminoacid sequence as that shown in FIG. 7 (SEQ ID NO:32). Also provided is aLM609 grafted antibody or functional fragment thereof. The antibody isgenerated from the above heavy and light chain polypeptides orfunctional fragments thereof and exhibits selective binding affinity toα_(v)β₃.

[0080] The invention provides an enhanced LM609 grafted antibodyexhibiting selective binding affinity to α_(v)β₃. The enhanced LM609grafted antibody contains at least one amino acid substitution in one ormore CDRs of a LM609 grafted heavy chain variable region polypeptide ora LM609 grafted light chain variable region polypeptide, wherein theα_(v)β₃ binding affinity of the enhanced LM609 grafted antibody ismaintained or enhanced.

[0081] To identify enhanced LM609 grafted antibodies, a library ofmodified LM609 grafted antibodies was generated as described above andin Example VI. Initially, LM609 CDRs were identified and selected tointroduce single amino acid substitutions. Utilizing the numberingsystem of Kabat et al., supra, the CDR residues selected for mutagenesiswere V_(H) CDR1 Gly-Phe-Thr-Phe-Ser-Ser-Tyr-Asp-Met-Ser (SEQ ID NO:34)(Gly²⁶-Ser³⁵); V_(H) CDR2 Trp-Val-Ala-Lys-Val-Ser-Ser-Gly-Gly-Gly (SEQID NO:36) and Ser-Thr-Tyr-Tyr-Leu-Asp-Thr-Val-Gln-Gly (SEQ ID NO:33)(Trp⁴⁷-Gly⁶⁵); V_(H) CDR3 Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Tyr (SEQID NO:40) (Ala⁹³-Tyr¹⁰²); V_(L) CDR1Gln-Ala-Ser-Gln-Ser-Ile-Ser-Asn-His-Leu-His-Trp-Tyr (SEQ ID NO:42)(Gln²⁴-Tyr³⁶); V_(L) CDR2 Leu-Leu-Ile-Arg-Tyr-Arg-Ser-Gln-Ser-Ile-Ser(SEQ ID NO:44) (Leu⁴⁶-Ser⁵⁶); and V_(L) CDR3Gln-Gln-Ser-Gly-Ser-Trp-Pro-His-Thr (SEQ ID NO:46) (Gln⁸⁹-Thr⁹⁷).

[0082] The nucleotide sequences encoding the CDR residues selected formutagenesis were V_(H) CDR1 GGA TTC ACC TTC AGT AGC TAT GAC ATG TCT (SEQID NO:33); V_(H) CDR2 TGG GTC GCA AAA GTT AGT AGT GGT GGT GGT (SEQ IDNO:35) and AGC ACC TAC TAT TTA GAC ACT GTG CAG GGC (SEQ ID NO:37);V_(H) CDR3 GCA AGA CAT AAC TAC GGC AGT TTT GCT TAC (SEQ ID NO:39);V_(L) CDR1 CAG GCC AGC CAA AGT ATT AGC AAC CAC CTA CAC TGG TAT (SEQ IDNO:41); V_(L) CDR2 CTT CTC ATC CGT TAT CGT TCC CAG TCC ATC TCT (SEQ IDNO:43); and V_(L) CDR3 CAA CAG AGT GGC AGC TGG CCT CAC ACG (SEQ IDNO:45).

[0083] Single amino acid substitutions can be introduced into the CDRsof an LM609 grafted antibody to generate a population of modified LM609grafted antibodies. For example, every amino acid in one or more CDRscan be mutated to any or all amino acids to generate a population ofmodified LM609 grafted antibodies and the population screened forα_(v)β₃ binding activity. Although this population is generated bymutating amino acids in CDRS, populations can also be constructed wherechanges are made in the framework region residues or in both the CDRsand the framework. Such mutations in the variable regions can be madeseparately, in combination, or step-wise. Thus, the invention alsoprovides for an enhanced LM609 grafted antibody, where the amino acidsubstitution is in the CDR or in the framework region.

[0084] The invention additionally provides an enhanced LM609 graftedantibody exhibiting enhanced binding affinity. Enhanced LM609 graftedantibodies exhibiting enhanced binding affinity include those containingat least one of the following CDRs having single amino acidsubstitutions: a V_(H) CDR1 selected from the group consisting ofGly-Thr-Thr-Phe-Ser-Ser-Tyr-Asp-Met-Ser (SEQ ID NO:48),Gly-Phe-Thr-Trp-Ser-Ser-Tyr-Asp-Met-Ser (SEQ ID NO:50) andGly-Phe-Thr-Phe-Leu-Ser-Tyr-Asp-Met-Ser (SEQ ID NO:52); a V_(H) CDR2selected from the group consisting ofTrp-Val-Ala-Lys-Val-Lys-Ser-Gly-Gly-Gly (SEQ ID NO:54),Ser-Thr-Tyr-Tyr-Pro-Asp-Thr-Val-Gln-Gly (SEQ ID NO:56) andSer-Thr-Tyr-Tyr-Leu-Asp-Thr-Val-Glu-Gly (SEQ ID NO:58); a V_(H) CDR3selected from the group consisting ofAla-Arg-His-Asn-His-Gly-Ser-Phe-Ala-Tyr (SEQ ID NO:60),Ala-Arg-His-Asn-Tyr-Gly-Ser-Tyr-Ala-Tyr (SEQ ID NO:62),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Asp-Tyr (SEQ ID NO:64),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Tyr-Tyr (SEQ ID NO:66),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Ser (SEQ ID NO:68),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Thr (SEQ ID NO:70),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Asp (SEQ ID NO:72),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Glu (SEQ ID NO:74),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Met (SEQ ID NO:76),Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Gly (SEQ ID NO:78) andAla-Arg-His-Asn-Tyr-Gly-Ser-Phe-Ala-Ala (SEQ ID NO:8O); the V_(L) CDR1Gln-Ala-Ser-Gln-Ser-Ile-Ser-Asn-Phe-Leu-His-Trp-Tyr (SEQ ID NO:82); theV_(L) CDR2 Leu-Leu-Ile-Arg-Tyr-Ser-Ser-Gln-Ser-Ile-Ser (SEQ ID NO:84);and a V_(L) CDR3 selected from the group consisting ofGln-Gln-Ser-Asn-Ser-Trp-Pro-His-Thr (SEQ ID NO:86),Gln-Gln-Ser-Thr-Ser-Trp-Pro-His-Thr (SEQ ID NO:88),Gln-Gln-Ser-Gly-Ser-Trp-Pro-Leu-Thr (SEQ ID NO:90) andGln-Gln-Ser-Gly-Ser-Trp-Pro-Gln-Thr (SEQ ID NO:92)

[0085] The nucleotide sequences encoding the CDRs having single aminoacid substitutions were V_(H) CDR1 GGA ACT ACC TTC AGT AGC TAT GAC ATGTCT (SEQ ID NO:47), GGA TTC ACC TGG AGT AGC TAT GAC ATG TCT (SEQ IDNO:49), and GGA TTC ACC TTC CTG AGC TAT GAC ATG TCT (SEQ ID NO:51);V_(H) CDR2 TGG GTC GCA AAA GTT AAA AGT GGT GGT GGT (SEQ ID NO:53), AGCACC TAC TAT CCT GAC ACT GTG CAG GGC (SEQ ID NO:55), and AGC ACC TAC TATTTA GAC ACT GTG GAG GGC (SEQ ID NO:57); V_(H) CDR3 GCA AGA CAT AAC CATGGC AGT TTT GCT TAC (SEQ ID NO:59), GCA AGA CAT AAC TAC GGC AGT TAT GCTTAC (SEQ ID NO:61), GCA AGA CAT AAC TAC GGC AGT TTT GAT TAC (SEQ IDNO:63), GCA AGA CAT AAC TAC GGC AGT TTT TAT TAC (SEQ ID NO:65), GCA AGACAT AAC TAC GGC AGT TTT GCT TCT (SEQ ID NO:67), GCA AGA CAT AAC TAC GGCAGT TTT GCT ACT (SEQ ID NO:69), GCA AGA CAT AAC TAC GGC AGT TTT GCT GAT(SEQ ID NO:71), GCA AGA CAT AAC TAC GGC AGT TTT GCT GAG (SEQ ID NO:73),GCA AGA CAT AAC TAC GGC AGT TTT GCT ATG (SEQ ID NO:75), GCA AGA CAT AACTAC GGC AGT TTT GCT GGG (SEQ ID NO:77), and GCA AGA CAT AAC TAC GGC AGTTTT GCT GCT (SEQ ID NO:79); V_(L) CDR1 CAG GCC AGC CAA AGT ATT AGC AACTTT CTA CAC TGG TAT (SEQ ID NO:81); V_(L) CDR2 CTT CTC ATC CGT TAT TCTTCC CAG TCC ATC TCT (SEQ ID NO:83); and V_(L) CDR3 GAA CAG AGT AAT AGCTGG CCT CAC ACG (SEQ ID NO:85), CAA CAG AGT ACT AGC TGG CCT CAC ACG (SEQID NO:87), CAA CAG AGT GGC AGC TGG CCT CTG ACG (SEQ ID NO:89) and CAACAG AGT GGC AGC TGG CCT CAG ACG (SEQ ID NO:91).

[0086] Enhanced LM609 grafted antibodies having CDRs with single aminoacid substitutions and higher affinity binding than the parent LM609grafted antibody can also be identified, where the corresponding aminoacid mutations are combined to generate new modified LM609 graftedantibodies. Identification is performed by screening for α_(v)β₃ bindingactivity. In some combinations, the LM609 grafted antibody will compriseat least one CDR having two or more amino acid substitutions. Theinvention provides an enhanced LM609 grafted antibody containing atleast one of the following CDRs containing multiple amino acidsubstitutions: a V_(H) CDR3 selected from the group consisting ofAla-Arg-His-Asn-His-Gly-Ser-Phe-Ala-Ser (SEQ ID NO:94);Ala-Arg-His-Asn-His-Gly-Ser-Phe-Tyr-Ser (SEQ ID NO:96);Ala-Arg-His-Asn-Tyr-Gly-Ser-Phe-Tyr-Glu (SEQ ID NO:98); andAla-Arg-His-Asn-Tyr-Gly-Ser-Phe-Tyr-Ser (SEQ ID NO:100).

[0087] The nucleotide sequences encoding the CDRS having multiple aminoacid substitutions were V_(H) CDR3 GCA AGA CAT AAC CAT GCC AGT TTT GCTTCT (SEQ ID NO:93), GCA AGA CAT AAC CAT GGC AGT TTT TAT TCT (SEQ IDNO:95), GCA AGA CAT AAC TAC GGC AGT TTT TAT GAG (SEQ ID NO:97), and GCAAGA CAT AAC TAC GGC AGT TTT TAT TCT (SEQ ID NO:99).

[0088] The invention also provides an enhanced LM609 grafted antibodyexhibiting selective binding affinity to α_(v)β₃, wherein the enhancedLM609 grafted antibody contains at least one amino acid substitution intwo or more CDRs of a LM609 grafted heavy chain variable regionpolypeptide or a LM609 grafted light chain variable region polypeptide.

[0089] An enhanced LM609 grafted antibody containing at least one aminoacid substitution in two or more CDRs of a LM609 grafted heavy chainvariable region polypeptide or a LM609 grafted light chain variableregion polypeptide can include an LM609 grafted antibody containing thecombination of CDRs selected from the group consisting of: the V_(L)CDR1 SEQ ID NO:57 and the V_(H) CDR3 SEQ ID NO:50; the V_(L) CDR1 SEQ IDNO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3 SEQ ID NO:50; theV_(L) CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3SEQ ID NO:52; the V_(L) CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44and the V_(H) CDR3 SEQ ID NO:51; the V_(L) CDR1 SEQ ID NO:57 and theV_(H) CDR3 SEQ ID NO:52; the V_(L) CDR3 SEQ ID NO:59, the V_(H) CDR2 SEQID NO:44 and the V_(H) CDR3 SEQ ID NO:50; the V_(L) CDR3 SEQ ID NO:61and V_(H) CDR3 SEQ ID NO:50; and the V_(L) CDR3 SEQ ID NO:61, the V_(H)CDR2 SEQ ID NO:44 and V_(H) CDR3 SEQ ID NO:50.

[0090] In addition to enhanced LM609 grafted antibodies containing twoor more CDRs having single amino acid substitutions, the invention alsoprovides enhanced LM609 grafted antibodies wherein at least one of theCDRs has two or more amino acid substitutions.

[0091] Enhanced LM609 grafted antibodies having at least one CDR withtwo or more amino acid substitutions can include those containing thecombination of CDRs selected from the group consisting of: the V_(L)CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3 SEQ IDNO:63; the V_(L) CDR3 SEQ ID NO:61, the V_(H) CDR2 SEQ ID NO:44 and theV_(H) CDR3 SEQ ID NO:63; the V_(L) CDR3 SEQ ID NO:61, the V_(H) CDR2 SEQID NO:44 and the V_(H) CDR3 SEQ ID NO:64; the V_(L) CDR3 SEQ ID NO:61and the V_(H) CDR3 SEQ ID NO:63; the V_(L) CDR3 SEQ ID NO:61 and theV_(H) CDR3 SEQ ID NO:65; and the V_(L) CDR3 SEQ ID NO:61, the V_(H) CDR2SEQ ID NO:44 and the V_(H) CDR3 SEQ ID NO:66.

[0092] The invention additionally provides a high affinity LM609 graftedantibody exhibiting selective binding affinity to α_(v)β₃. The highaffinity LM609 grafted antibody contains at least one amino acidsubstitution in one or more CDRs of a LM609 grafted heavy chain variableregion polypeptide or a LM609 grafted light chain variable regionpolypeptide, wherein the α_(v)β₃ binding affinity of the high affinityLM609 grafted antibody is enhanced.

[0093] High affinity antibodies can include those containing thecombination of CDRs selected from the group consisting of: the V_(L)CDR1 SEQ ID NO:57 and the V_(H) CDR3 SEQ ID NO:50; the V_(L) CDR1 SEQ IDNO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3 SEQ ID NO:50; theV_(L) CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3SEQ ID NO:52; the V_(L) CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44and the V_(H) CDR3 SEQ ID NO:51; the V_(L) CDR1 SEQ ID NO:57 and theV_(H) CDR3 SEQ ID NO:52; the V_(L) CDR3 SEQ ID NO:59, the V_(H) CDR2 SEQID NO:44 and the V_(H) CDR3 SEQ ID NO:50; the V_(L) CDR3 SEQ ID NO:61,the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3 SEQ ID NO:63; the V_(L)CDR3 SEQ ID NO:61 and V_(H) CDR3 SEQ ID NO:50; the V_(L) CDR3 SEQ IDNO:61, the V_(H) CDR2 SEQ ID NO:44 and V_(H) CDR3 SEQ ID NO:50; theV_(L) CDR1 SEQ ID NO:57, the V_(H) CDR2 SEQ ID NO:44 and the V_(H) CDR3SEQ ID NO:63; the V_(L) CDR3 SEQ ID NO:61, the V_(H) CDR2 SEQ ID NO:44and the V_(H) CDR3 SEQ ID NO:64; the V_(L) CDR3 SEQ ID NO:61 and theV_(H) CDR3 SEQ ID NO:63; the V_(L) CDR3 SEQ ID NO: 61 and the V_(H) CDR3SEQ ID NO:65; and the V_(L) CDR3 SEQ ID NO:61, the V_(H) CDR2 SEQ IDNO:44 and the V_(H) CDR3 SEQ ID NO:66.

[0094] The invention additionally provides a nucleic acid encoding anenhanced LM609 grafted antibody exhibiting selective binding affinity toα_(v)β₃. The enhanced LM609 grafted antibody encoded by the nucleic acidcontains at least one amino acid substitution in one or more CDRs of aLM609 grafted heavy chain variable region polypeptide or a LM609 graftedlight chain variable region polypeptide, wherein the α_(v)β₃ bindingaffinity of the enhanced LM609 grafted antibody is maintained orenhanced.

[0095] The invention further provides a nucleic acid encoding a highaffinity LM609 grafted antibody exhibiting selective binding affinity toα_(v)β₃. The high affinity LM609 grafted antibody encoded by the nucleicacid contains at least one amino acid substitution in one or more CDRsof a LM609 grafted heavy chain variable region polypeptide or a LM609grafted light chain variable region polypeptide, wherein the α_(v)β₃binding affinity of the high affinity LM609 grafted antibody isenhanced.

[0096] The invention provides a nucleic acid encoding a heavy chainpolypeptide for monoclonal antibody LM609 or functional fragmentthereof. Also provided is a nucleic acid encoding a light chainpolypeptide for monoclonal antibody LM609 or a functional fragmentthereof. The nucleic acids consist of substantially the same heavy orlight chain variable region nucleotide sequences as that shown in FIGS.2A and 2B (SEQ ID NOS:5 and 7, respectively) or a fragment thereof.

[0097] As described previously, monoclonal antibody LM609 has been shownin the art to have binding activity to the integrin α_(v)β₃. Althoughspecificity can in principle be generated towards essentially anytarget, LM609 is an integrin inhibitory antibody that exhibitssubstantial specificity and inhibitory activity to a single memberwithin an integrin family. In this case, LM609 exhibits substantialspecificity and inhibitory activity to the α_(v)β₃ integrin within theβ₃ family. The amino acid or nucleotide sequence of monoclonal antibodyLM609 has never been previously isolated and characterized.

[0098] The isolation and characterization of LM609 encoding nucleicacids was performed by techniques known to those skilled in the art andwhich are described further below in the Examples. Briefly, cDNA fromhybridoma LM609 was generated and used as the source for which toisolate LM609 encoding nucleic acids. Isolation was performed by firstdetermining the N-terminal amino acid sequence for each of the heavy andlight chain polypeptides and then amplifying by PCR the antibodyencoding sequences from the cDNA. The 5′ primers were reverse translatedto correspond to the newly determined N-terminal amino acid sequenceswhereas the 3′ primers corresponded to sequences substantially similarto antibody constant region sequences. Amplification and cloning of theproducts resulted in the isolation of the nucleic acids encoding heavyand light chains of LM609.

[0099] The nucleotide sequences of the LM609 heavy and light chainvariable region sequences are shown in FIGS. 2A and 2B, respectively.These sequences correspond substantially to those that encode thevariable region heavy and light chain polypeptides of LM609. As with theVitaxin nucleic acids, these LM609 nucleic acids are intended to includeboth sense and anti-sense strands of the LM609 encoding sequences.Single- and double-stranded nucleic acids are also include as well asnon-coding portions of the nucleic acid such as introns, 5′- and3′-untranslated regions and regulatory sequences of the gene forexample.

[0100] As shown in FIG. 2A, the LM609 heavy chain variable regionpolypeptide is encoded by a nucleic acid of about 351 nucleotides inlength which begins at the amino terminal Glu1 residue of the variableregion through to Ala 117. The murine LM609 antibody heavy chain has anIgG2a constant region. Shown in FIG. 2B is the LM609 light chainvariable region polypeptide which is encoded by a nucleic acid of about321 nucleotides in length which begins at the amino terminal Asp1residue of the variable region through to Lys 107. In the functionalantibody, LM609 has a kappa light chain constant region.

[0101] As with the Vitaxin nucleic acids, minor modifications of theseLM609 nucleotide sequences are intended to be included as heavy andlight chain LM609 encoding nucleic acids. Such minor modifications areincluded within the nucleic acids encoding LM609 heavy and light chainpolypeptides so long as the nucleic acids or encoded polypeptides retainsome or all of their function as described.

[0102] Thus, the invention also provides a nucleic acid encoding a LM609heavy chain or functional fragment wherein the nucleic acid encodessubstantially the same variable region amino acid sequence of monoclonalantibody LM609 as that shown in FIG. 2A (SEQ ID NO:6) or a fragmentthereof. Similarly, the invention also provides a nucleic acid encodinga LM609 light chain or functional fragment wherein the nucleic acidencodes substantially the same variable region amino acid sequence ofmonoclonal antibody LM609 as that shown in FIG. 2B (SEQ ID NO:8) or afragment thereof.

[0103] The invention further provides fragments of LM609 heavy and lightchain encoding nucleic acids wherein such fragments consistsubstantially of the same nucleotide or amino acid sequence as thevariable region of LM609 heavy or light chain polypeptides. The variableregion of the LM609 heavy chain polypeptide consists essentially ofnucleotides 1-351 and of amino acid residues Glu1 to Ala117 of FIG. 2A(SEQ ID NOS:5 and 6, respectively). The variable region of the LM609light chain polypeptide consists essentially of nucleotides 1-321 and ofamino acid residues Asp1 to Lys107 of FIG. 2B (SEQ ID NOS:7 and 8,respectively). The termini of such variable region encoding nucleicacids is not critical so long as the intended purpose and functionremains the same. Such intended purposes and functions include, forexample, use for the production of recombinant polypeptides or ashybridization probes for heavy and light chain variable regionsequences.

[0104] Fragments additional to the variable region nucleic acidfragments are provided as well. Such fragments include, for example,nucleic acids consisting substantially of the same nucleotide sequenceas a CDR of a LM609 heavy or light chain polypeptide. Sequencescorresponding to the LM609 CDRs include, for example, those regionswithin the variable region which are defined by Kabat et al., supra,and/or those regions within the variable regions which are defined byChothia et al., supra, as well as those regions defined by MacCallum etal., supra. The LM609 CDR fragments for each of the above definitionscorrespond to the nucleotides set forth below in Table 4. The nucleotidesequence numbering is taken from the primary sequence shown in FIGS. 2Aand 2B (SEQ ID NOS:5 and 7) and conforms to the definitions previouslyset forth in Table 1. TABLE 4 LM609 CDR Nucleotide Residues KabatChothia MacCallum V_(H) CDR1  91-105 76-96  88-105 V_(H) CDR2 148-198157-168 139-177 V_(H) CDR3 295-318 298-315 288-315 V_(L) CDR1  70-10276-96  88-108 V_(L) CDR2 148-168 148-156 136-165 V_(L) CDR3 265-291271-288 265-288

[0105] Similarly, the LM609 CDR fragments for each of the abovedefinitions correspond to the amino acid residues set forth below inTable 5. The amino acid residue numbering is taken from the primarysequence shown in FIGS. 2A and 2B (SEQ ID NOS:6 and 8) and conforms tothe definitions set forth in Table 1. TABLE 5 LM609 CDR Amino AcidResidues Kabat Chothia MacCallum V_(H) CDR1 Ser31-Ser35 Gly26-Tyr32Ser30-Ser35 V_(H) CDR2 Lys50-GlyG6 Ser53-Gly56 Trp47-Tyr59 V_(H) CDR3His99-Tyr106 Asn100-Ala105 Ala97-Ala105 V_(L) CDR1 Gln24-His34Ser26-His32 Ser30-Tyr36 V_(L) CDR2 Tyr50-Ser56 Tyr50-Ser52 Leu46-Ile55V_(L) CDR3 Gln39-Thr97 Ser91-His96 Gln89-His96

[0106] Nucleic acids encoding LM609 heavy and light chain polypeptidesand fragments thereof are useful for a variety of diagnostic andtherapeutic purposes. For example, the LM609 nucleic acids can be usedto produce recombinant LM609 antibodies and functional fragments thereofhaving binding specificity and inhibitory activity against the integrinα_(v)β₃. The antibody and functional fragments thereof can be used todetermine the presence or absence of α_(v)β₃ in a sample to diagnose thesusceptibility or occurrence of an α_(v)β₃-mediated disease.Alternatively, the recombinant LM609 antibodies and functional fragmentsthereof can be used for the therapeutic treatment of α_(v)β₃-mediateddiseases or pathological state. As with Vitaxin, recombinant LM609 andfunctional fragments thereof can be used to inhibit the binding activityor other functional activities of α_(v)β₃ that are necessary forprogression of the α_(v)β₃-mediated disease or pathological state.

[0107] The LM609 nucleic acids of the invention can also be used tomodel functional equivalents of the encoded heavy and light chainpolypeptides. Such functional equivalents can include, for example,synthetic analogues or mimics of the encoded polypeptides or functionalfragments thereof. A specific example would include peptide mimetics ofthe LM609 CDRs that retain some or substantially the same binding orinhibitory activity of LM609. Additionally, the LM609 encoding nucleicacids can be used to engineer and produce nucleic acids which encodemodified forms or derivatives of the antibody LM609, its heavy and lightchain polypeptides and functional fragments thereof. As describedpreviously, such modified forms or derivatives include, for example,non-mouse antibodies, their corresponding heavy and light chainpolypeptides and functional fragments thereof which exhibitsubstantially the same binding and inhibitory activity as LM609.

[0108] The invention also provides a method of treating anα_(v)β₃-mediated disease. The method consists of administering aneffective amount of Vitaxin, a LM609 grafted antibody, an enhancedantibody thereof, or a functional fragment thereof under conditionswhich allow binding to α_(v)β₃. Also provided is a method of inhibitinga function of α_(v)β₃. The method consists of contacting α_(v)β₃ withVitaxin, a LM609 grafted antibody or a functional fragment thereof underconditions which allow binding to α_(v)β₃.

[0109] As described previously, Vitaxin and LM609 grafted antibodies aremonoclonal antibodies which exhibit essentially all of the bindingcharacteristics as does its parental CDR-donor antibody LM609. Thesecharacteristics include, for example, significant binding specificityand affinity for the integrin α_(v)β₃. The Examples below demonstratethese binding properties and further show that the binding of suchantibodies to α_(v)β₃ inhibits α_(v)β₃ ligand binding and function.Thus, Vitaxin and LM609 grafted antibodies are useful for a largevariety of diagnostic and therapeutic purposes directed to theinhibition of α_(v)β₃ function.

[0110] The integrin α_(v)β₃ functions in numerous cell adhesion andmigration associated events. As such, the dysfunction or dysregulationof this integrin, its function, or of cells expressing this integrin, isassociated with a large number of diseases and pathological conditions.The inhibition α_(v)β₃ binding or function can therefore be used totreat or reduce the severity of such α_(v)β₃-mediated pathologicalconditions. Described below are examples of several pathologicalconditions mediated by α_(v)β₃ since the inhibition of at least thisintegrin reduces the severity of the condition. These examples areintended to be representative and as such are not inclusive of allα_(v)β₃-mediated diseases. For example, there are numerous pathologicalconditions additional to those discussed below which exhibit thedysregulation of α_(v)β₃ binding, function or the dysregulation of cellsexpressing this integrin and in which the pathological condition can bereduced, or will be found to be reduced, by inhibiting the bindingα_(v)β₃. Such pathological conditions which exhibit this criteria, areintended to be included within the definition of the term as usedherein.

[0111] Angiogenesis, or neovascularization, is the process where newblood vessels form from pre-existing vessels within a tissue. Asdescribed further below, this process is mediated by endothelial cellsexpressing α_(v)β₃ and inhibition of at least this integrin, inhibitsnew vessel growth. There are a variety of pathological conditions thatrequire new blood vessel formation or tissue neovascularization andinhibition of this process inhibits the pathological condition. As such,pathological conditions that require neovascularization for growth ormaintenance are considered to be α_(v)β₃-mediated diseases. The extentof treatment, or reduction in severity, of these diseases will thereforedepend on the extent of inhibition of neovascularization. Theseα_(v)β₃-mediated diseases include, for example, inflammatory disorderssuch as immune and non-immune inflammation, chronic articularrheumatism, psoriasis, disorders associated with inappropriate orinopportune invasion of vessels such as diabetic retinopathy,neovascular glaucoma and capillary proliferation in atheroscleroticplaques as well as cancer disorders. Such cancer disorders can include,for example, solid tumors, tumor metastasis, angiofibromas, retrolental,fibroplasia, hemangiomas, Kaposi's sarcoma and other cancers whichrequire neovascularization to support tumor growth. Additional diseaseswhich are considered angiogenic include psoriasis and rheumatoidarthritis as well as retinal diseases such as macular degeneration.Diseases other than those requiring new blood vessels which areα_(v)β₃-mediated diseases include, for example, restenosis andosteoporosis.

[0112] Treatment of the α_(v)β₃-mediated diseases can be performed byadministering an effective amount of Vitaxin, a LM609 grafted antibody,an enhanced antibody thereof, or a functional fragment thereof so as tobind to α_(v)β₃ and inhibit its function. Administration can beperformed using a variety of methods known in the art. The choice ofmethod will depend on the specific α_(v)β₃-mediated disease and caninclude, for example, the in vivo, in situ and ex vivo administration ofVitaxin, a LM609 grafted antibody or functional fragment thereof, tocells, tissues, organs, and organisms. Moreover, such antibodies orfunctional fragments can be administered to an individual exhibiting orat risk of exhibiting an α_(v)β₃-mediated disease. Definite clinicaldiagnosis of an α_(v)β₃-mediated disease warrants the administration ofVitaxin, a LM609 grafted antibody or a functional fragment thereof.Prophylactic applications are warranted in diseases where theα_(v)β₃-mediated disease mechanisms precede the onset of overt clinicaldisease. Thus, individuals with familial history of disease andpredicted to be at risk by reliable prognostic indicators can be treatedprophylactically to interdict α_(v)β₃-mediated mechanisms prior to theironset.

[0113] Vitaxin, a LM609 grafted antibody, an enhanced antibody thereof,or functional fragments thereof can be administered in a variety offormulations and pharmaceutically acceptable media for the effectivetreatment or reduction in the severity of an α_(v)β₃-mediated disease.Such formulations and pharmaceutically acceptable medias are well knownto those skilled in the art. Additionally, Vitaxin, a LM609 graftedantibody or functional fragments thereof can be administered with othercompositions which can enhance or supplement the treatment or reductionin severity of an α_(v)β₃-mediated disease. For example, thecoadministration of Vitaxin or a LM609 grafted antibody to inhibittumor-induced neovascularization and a chemotherapeutic drug to directlyinhibit tumor growth is one specific case where the administration ofother compositions can enhance or supplement the treatment of anα_(v)β₃-mediated disease.

[0114] Vitaxin, a LM609 grafted antibody or functional fragments areadministered by conventional methods, in dosages which are sufficient tocause the inhibition of α_(v)β₃ integrin binding at the sight of thepathology. Inhibition can be measured by a variety of methods known inthe art such as in situ immunohistochemistry for the prevalence ofα_(v)β₃ containing cells at the site of the pathology as well asinclude, for example, the observed reduction in the severity of thesymptoms of the α_(v)β₃-mediated disease.

[0115] In vivo modes of administration can include intraperitoneal,intravenous and subcutaneous administration of Vitaxin, a LM609 graftedantibody or a functional fragment thereof. Dosages for antibodytherapeutics are known or can be routinely determined by those skilledin the art. For example, such dosages are typically administered so asto achieve a plasma concentration from about 0.01 μg/ml to about 100μg/ml, preferably about 1-5 μg/ml and more preferably about 5 μg/ml. Interms of amount per body weight, these dosages typically correspond toabout 0.1-300 mg/kg, preferably about 0.2-200 mg/kg and more preferablyabout 0.5-20 mg/kg. Depending on the need, dosages can be administeredonce or multiple times over the course of the treatment. Generally, thedosage will vary with the age, condition, sex and extent of theα_(v)β₃-mediated pathology of the subject and should not be so high asto cause adverse side effects. Moreover, dosages can also be modulatedby the physician during the course of the treatment to either enhancethe treatment or reduce the potential development of side effects. Suchprocedures are known and routinely performed by those skilled in theart.

[0116] The specificity and inhibitory activity of Vitaxin, LM609 graftedantibodies, an enhanced antibody thereof and functional fragmentsthereof allow for the therapeutic treatment of numerous α_(v)β₃-mediateddiseases. Such diseases include, for example, pathological conditionsrequiring neovascularization such as tumor growth, and psoriasis as wellas those directly mediated by α_(v)β₃ such as restenosis andosteoporosis. Thus, the invention provides methods as well as Vitaxinand LM609 grafted antibody containing compositions for the treatment ofsuch diseases.

[0117] Throughout this application various publications are referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

[0118] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Isolation and Characterization of LM609 Encoding Nucleic Acids

[0119] This Example shows the cloning and sequence determination ofLM609 encoding nucleic acids.

[0120] LM609 is directed against the human vitronectin receptor,integrin α_(v)β₃. α_(v)β₃ is highly upregulated in melanoma,glioblastoma, and mammary carcinoma and plays a role in theproliferation of M21 melanoma cells both in vitro and in vivo. α_(v)β₃also plays a role in angiogenesis, restenosis and the formation ofgranulation tissue in cutaneous wounds. LM609 has been shown to inhibitthe adhesion of M21 cells to vitronectin as well as preventproliferation of M21 cells in vitro. Thus, grafting of LM609 couldresult in a clinically valuable therapeutic agent.

[0121] cDNA Synthesis of LM609 Variable Regions: For cDNA synthesis,total RNA was prepared from 10⁸ LM609 hybridoma cells using amodification of the method described by Chomczynski and Sacchi(Chomczynski and Sacchi, Analyt. Biochem. 162:156 (1987)). LM609variable (V) region genes were cloned by reversetranscription-polymerase chain reaction (RT-PCR) and cDNA wassynthesized using BRL Superscript kit. Briefly, 5 μg of total cellularRNA, 650 ng oligo dT and H₂O were brought to a total volume of 55 μl.The sample was heated to 70° C. for 10 min and chilled on ice. Reactionbuffer was added and the mixture brought to 10 mM DTT and 1 mM dNTPs andheated at 37° C. for 2 minutes. 5 μl (1000 units) reverse transcriptasewas added and incubated at 37° C. for 1 hour and then chilled on ice.

[0122] All oligonucleotides were synthesized by β-cyanoethylphosphoramidite chemistry on an ABI 394 DNA synthesizer.Oligonucleotides used for PCR amplification and routine site-directedmutagenesis were purified using oligonucleotide purification cartridges(Applied Biosystems, Foster City, Calif.). Forward PCR primers weredesigned from N-terminal protein sequence data generated from purifiedLM609 antibody. The forward PCR primers contained sequences coding forthe first six amino acids in each antibody variable chain (proteinsequenced at San Diego State University). The sequence of the lightchain forward PCR primer (997) was 5′-GCC CAA CCA GCC ATG GCC GAT ATTGTG CTA ACT CAG-3′ (SEQ ID NO:19) whereas the light chain reverse PCRprimer (734) was 5′-AC AGT TGG TGC AGC ATC AGC-3′ (SEQ ID NO:20) used.This reverse primer corresponds to mouse light chain kappa amino acidresidues 109-115. The sequence of the heavy chain forward PCR primer(998) was 5′-ACC CCT GTG GCA AAA GCC GAA GTG CAG CTG GTG GAG-3′ (SEQ IDNO:21). Heavy chain reverse PCR primer 733: 5′-GA TGG GGG TGT CGT TTTGGC-3′ SEQ ID NO:22). The PCR primers also contain regions of homologywith specific sequences within the immunoexpression vector.

[0123] V_(L) and V_(H) chains were amplified in two separate 50 μlreaction mixtures containing 2 μl of the cDNA-RNA heteroduplex, 66.6 mMTris-HCl pH 8.8, 1.5 mM MgCl₂, 0.2 mM of each four dNTPs, 10 mM2-mercaptoethanol, 0.25 units Taq polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and 50 pmoles each of primers 997 and 734 and 998and 733, respectively. The mixtures were overlaid with mineral oil andcycled for two rounds of PCR with each cycle consisting of 30 seconds at94° C. (denature), 30 seconds at 50° C. (anneal), and 30 seconds at 72°C. (synthesis). This reaction was immediately followed by 30 cycles ofPCR consisting of 30 seconds at 94° C. (denature), 30 seconds at 55° C.(anneal), and 30 seconds at 72° C. (synthesis) followed by a finalsynthesis reaction for 5 minutes at 72° C. The reaction products werepooled, extracted with CHCl₃ and ethanol precipitated.

[0124] Amplified products were resuspended in 20 μl TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 8.0) and electrophoresed on a 5% polyacrylamidegel. Bands migrating at expected molecular weights of V_(H) and V_(L)were excised, chemically eluted from the gel slice, extracted withorganic solvents and ethanol precipitated.

[0125] Cloning of amplified V_(H) and V_(L) genes into M13 phageimmunoexpression vector: The amplified V region gene products weresequentially cloned into the phage immunoexpression vector byhybridization mutagenesis (Near, R. Biotechniques 12:88 (1992); Yeltonet al., J. Immunol. 155:1994-2003 (1995)). Introduction of the amplifiedV_(L) and V_(H) sequences by hybridization mutagenesis positions theantibody sequences in frame with the regulatory elements contained inthe M13 vector required for efficient Fab expression. One advantage ofthis technique is that no restriction endonuclease sites need to beincorporated into the V_(L) or V_(H) gene sequences for cloning as isdone with conventional DNA ligation methods.

[0126] To perform the cloning, 400 ng each of the double-strandedamplified products were first phosphorylated with polynucleotide kinase.100 ng of the phosphorylated LM609 V_(L) product was mixed with 250 ngof uridinylated BS11 phage immunoexpression vector, denatured by heatingto 90° C. and annealed by gradual cooling to room temperature. BS11 isan M13 immunoexpression vector derived from M13 IX and encodes CH₁ ofmurine IgG1 and murine kappa light chain constant domain (Huse, W. D.In: Antibody Engineering: A Practical Guide, C. A. K. Borrebaeck, ed. W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).Nucleotide sequences included in the PCR amplification primers anneal tocomplementary sequences present in the single-stranded BS11 vector. Theannealed mixture was fully converted to a double-stranded molecule withT4 DNA polymerase plus dNTPs and ligated with T4 ligase. 1 μl of themutagenesis reaction was electroporated into E. coli strain DH10B,titered onto a lawn of XL-1 E. coli and incubated until plaques formed.Plaque lift assays were performed as described using goat anti-murinekappa chain antibody conjugated to alkaline phosphatase (Yelton et al,supra; Huse, W. D., supra). Fifteen murine light chain positive M13phage clones were isolated, pooled and used to prepare uridinylatedvector to serve as template for hybridization mutagenesis with the PCRamplified LM609 V_(H) product.

[0127] Clones expressing functional murine LM609 Fab were identified bybinding to purified α_(v)β₃ by ELISA. Briefly, Immulon II ELISA plateswere coated overnight with 1 μg/ml (100 ng/well) α_(v)β₃ and nonspecificsites blocked for two hours at 27° C. Soluble Fabs were prepared byisolating periplasmic fractions of cultures of E. coli strain MK30-3(Boehringer Mannheim Co.) infected with the Fab expressing M13 phageclones. Periplasm fractions were mixed with binding buffer 100 mM NaCl,50 mM Tris pH 7.4, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 0.02% NaN₃, 1mg/ml BSA and incubated with immobilized α_(v)β₃ for two hours at 27° C.Plates were washed with binding buffer and bound Fab detected with goatanti-murine kappa chain antibody conjugated to alkaline phosphatase.Four α_(v)β₃ reactive clones were identified: muLM609M13 12, 29, 31 and69. MuLM609M13 12 and 29 gave the strongest signals in the ELISA assay.DNA sequence analysis showed that clones muLM609M13 12, 31 and 69 allhad identical light chain sequence and confirmed the previouslydetermined N-terminal amino acid sequence of purified LM609 light chainpolypeptide. All four clones had identical V_(H) DNA sequence and alsoconfirmed the previously determined N-terminal amino acid sequence ofpurified LM609 heavy chain polypeptide.

[0128] To further characterize the binding activity of each clone,soluble Fab fractions were prepared from 50 ml cultures of E. colistrain MK30-3 infected with clones 12 and 29 and evaluated for bindingto α_(v)β₃ in a competitive ELISA with LM609 IgG. The results of thisELISA are shown in FIG. 3. Clone muLM609M13 12 was found to inhibitLM609 IgG binding (at LM609 IgG concentrations of 1 ng/ml and 5 ng/ml)to α_(v)β₃ in a concentration dependent manner at periplasm titersranging from neat to 1:80. Clone muLM609M13 12 was plaque purified andboth the V region heavy and light chain DNA sequences again determined.Complete DNA sequence of the final clone, muLM609M13 12-5, is shown inFIGS. 2A and 2B.

EXAMPLE II Construction of Vitaxin: A CDR Grafted LM609 FunctionalFragment

[0129] One goal of grafting antibodies is to preserve antibodyspecificity and affinity when substituting non-human CDRs into a humanantibody framework. Another goal is to minimize the introduction offoreign amino acid sequences so as to reduce the possible antigenicitywith a human host. This Example describes procedures for accomplishingboth of these goals by producing libraries of grafted antibodies whichrepresent all possible members which exhibit the highest affinities forthe desired antigen.

[0130] The above library was constructed in E. coli wherein the possibleCDR and framework changes were incorporated using codon-basedmutagenesis (Kristensson et al., In: Vaccines 95. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y. (1995); Rosok et al., J.Biol. Chem. (271:22611-22613 (1996)). Using these procedures, a librarywas constructed and a functionally active humanizedanti-α_(v)β₃-inhibitory antibody was identified.

[0131] For the construction of one grafted form of LM609, humanframework sequences showing the highest degree of identity to the murineLM609 V region gene sequences were selected for receiving the LM609CDRs. Human heavy chain V region M72 ′CL (HHC30Q, HC Subgroup 3, Kabatet al., supra) had 88% identity to frameworks 1, 2 and 3 of LM609 heavychain and human light chain V region LS1 ′CL (HKL312, Kappa subgroup 3,Kabat et al., supra) had 79% identity to frameworks 1, 2 and 3 of LM609light chain. Murine LM609 CDR sequences, as defined by Kabat et al.,supra were grafted onto the human frameworks. Residues predicted to beburied that might affect the structure and therefore the bindingproperties of the original murine combining site were taken intoconsideration when designing possible changes (Singer et al., supra;Padlan, E. A. Mol. Immunol. 28:489-498 (1991)). This analysis offramework residues considered to be important for preserving thespecificity and affinity of the combining site revealed only a fewdifferences. For example, in the heavy chain sequence, the predictedburied residues displayed 100% identity. Of particular note is thatArg16 in human heavy chain V region M72 ′CL is a relatively uncommonresidue among human chains. However, this residue was also found to bepresent in LM609 V_(H) and therefore was retained. Similarly, Arg19 inLM609 is a relatively rare residue among murine heavy chains but it isfound to occur in M72 ′CL and was therefore retained. In the light chainsequences, two nonidentical buried residues were identified betweenLM609 and LS1 ′CL framework regions at positions 49 and 87. These twopositions were therefore incorporated into the grafted antibody libraryas both human and murine alternatives.

[0132] Full-length grafted V region genes were synthesized by PCR usinglong overlapping oligonucleotides. Light chain oligonucleotidescontaining mixed amino acid residues at positions 49 and 87 weresynthesized as described in Glaser et al. (J. Immunol. 149:3903-3913(1992)) and as illustrated in the oligonucleotides represented as V_(L)oligo3 and V_(L) oligo4. (SEQ ID NOS:16 and 17, respectively). All longoligonucleotides were gel purified.

[0133] Grafted LM609 heavy and light chain V regions were constructed bymixing 5 overlapping oligonucleotides at equimolar concentrations, inthe presence of annealing PCR primers. The heavy chain oligonucleotidesmap to the following nucleotide positions: V_(H) oligonucleotide 1(V_(H) oligol), nucleotides (nt) 1-84; (SEQ ID NO: 9); V_(H) oligo2, nt70-153, (SEQ ID NO:10); V_(H) oligo3, nt 138-225 (SEQ ID NO:11); V_(H)oligo4, nt 211-291 (SEQ ID NO:12); V_(H) oligo5, nt 277-351 (SEQ IDNO:13). Similarly, the Vitaxin light chain oligonucleotides map to thefollowing nucleotide positions: V_(L) oligonucleotide 1 (V_(L) oligol),nucleotides (nt) 1-87; (SEQ ID NO:14); V_(L) oligo2, nt 73-144, (SEQ IDNO:15); V_(L) oligo3, nt 130-213 (SEQ ID NO:16); V_(L) oligo4, nt199-279 (SEQ ID NO:17); V_(L) oligo5, nt 265-321 (SEQ ID NO:18). Thenucleotide sequences of oligonucleotides used to construct grafted LM609heavy and light chain variable regions are shown in Table 6. Codonpositions 49 and 87 in V_(L) oligo3, and V_(L) oligo4 represent therandomized codons. The annealing primers contained at least 18nucleotide residues complementary to vector sequences for efficientannealing of the amplified V region product to the single-strandedvector. The annealed mixture was fully converted to a double-strandedmolecule with T4 DNA polymerase plus dNTPs and ligated with T4 ligase.TABLE 6 Oligonucleotides Used to Construct Grafted LM609 Heavy and LightChain Variable Regions CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTTGTGCAGCCTGGAAGGTC CCTGAGACTC SEQ ID NO:9 TCCTGTGCAG CCTCTGGATT CACC AACTTTTGCGACCCACTCCA GACCCTTGCC CGGAGCCTGG CGAACCCAAG ACATGTCATA SEQ ID NO:10GCTACTGAAG GTGAATCCAG AGGC TGGGTCGCAA AAGTTAGTAG TGGTGGTGGT AGCACCTACTATTTAGACAC TGTGCAGGGC SEQ ID NO:11 CGATTCACCA TCTCCAGAGA CAATAGTTGCACAGTAA TACACGGCTG TGTCCTCGGC TCTCAGAGAG TTCATTTGCA GGTATAGGGT SEQ IDNO:12 GTTCTTACTA TTCTCTCTGG A GTGTATTACT GTGCAAGACA TAACTACGGCAGTTTTGCTT ACTGGGGCCA AGGGACTACA SEQ ID NO:13 GTGACTGTTT CTAGTGAGATTGTGC TAACTCAGTC TCCAGCCACC CTGTCTCTCA GCCCAGGAGA AAGGGCGACT SEQ IDNO:14 CTTTCCTGCC AGGCCAGCCA AAGTATT GATGAGAAGC CTTGGGGCTT GACCAGGCCTTTGTTGATAC CAGTGTAGGT GGTTGCTAAT SEQ ID NO:15 ACTTTGGCTG GC CCAAGGCTTCTCATCWASTA TCGTTCCCAG TCCATCTCTG GCATCCCCGC CAGGTTCAGT SEQ ID NO:16GGCAGTGGAT CAGGGACAGA TTTC GCTGCCACTC TGTTGACAGW AATAGACTGC AAAATCTTCAGGCTCCAGAC TGGAGATAGT SEQ ID NO:17 GAGGGTGAAA TCTGTCCCTG A CAACAGAGTGGCAGCTGGCC TCACACGTTC GGAGGGGGGA CCAAGGTGGA AATTAAG SEQ ID NO:18

[0134] To generate the library, a portion of the mutagenesis reaction (1μl) was electroporated into E. coli strain DH10B (BRL), titered onto alawn of XL-1 (Stratagene, Inc.) and incubated until plaques formed.Replica filter lifts were prepared and plaques containing V_(H) genesequences were screened either by hybridization with adigoxigenin-labeled oligonucleotide complementary to LM609 heavy chainCDR 2 sequences or reactivity with 7F11-alkaline phosphatase conjugate,a monoclonal antibody raised against the decapeptide sequence Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser (SEQ ID NO:28) appended to the carboxyterminus of the vector CH₁ domain (Biosite, Inc., San Diego, Calif.).Fifty clones that were double-positive were pooled and used to prepareuridinylated template for hybridization mutagenesis with the amplifiedgrafted LM609 V_(L) product.

[0135] The mutagenesis reaction was performed as described above withthe V_(H) oligonucleotides except that the V_(L) oligonucleotides 1 to 5were employed (SEQ ID NOS:14 to 18, respectively). The reaction waselectroporated into E. coli strain DH10B and filter lifts probed witheither goat anti-human kappa chain antibody conjugated to alkalinephosphatase or a goat anti-human Fab antibody using an alkalinephosphatase conjugated rabbit anti-goat secondary reagent for detection.Positive clones co-expressing both V_(H) and V_(L) gene sequences wereselected (160 total) and used to infect E. coli strain MK30-3 forpreparing soluble Fab fragments.

[0136] The soluble Fab fragments were screened for binding to α_(v)β₃ inan ELISA assay. Four clones that were shown from the ELISA to stronglybind α_(v)β₃ were identified and further characterized. These cloneswere termed huLM609Ml3-34, 54, 55 and 145. All four clones were plaquepurified and three independent subclones from each clone was used toprepare Fab fragments for additional binding analysis to α_(v)β₃ byELISA.

[0137] In this additional ELISA, duplicate plates were coated withα_(v)β₃ ligand and incubated with the huLM609 periplasmic samples. Inone plate, bound huLM609 Fab was detected with goat anti-human kappachain antibody conjugated to alkaline phosphatase and in the other platebound huLM609 Fab was detected with 7F11-alkaline phosphatase conjugate,the monoclonal antibody recognizing the decapeptide tag. SubcloneshuLM609M13-34-1, 2 and 3 and huLM609M13-145-1, 2 and 3 all yieldeddouble positive signals indicating that the Fabs contain functionalV_(H) and V_(L) polypeptides. These results were confirmed in an ELISAassay on M21 cells, a cell line that expresses the integrin α_(v)β₃. DNAsequence analysis of subclones huLM609M13-34-3 and huLM609M13-145-3revealed mutations introduced into the library by errors due tooligonucleotide synthesis or by errors arising during PCR amplification.These mutations were corrected in clone huLM609M13-34-3 by site-directedmutagenesis. In the light chain sequence the following corrections weremade: His36 to Tyr36 and Lys18 to Arg18. In the heavy chain sequence thefollowing corrections were made: Glu1 to Gln1, Asn3 to Gln3, Leu11 toVal11. Additionally, during the construction of LM609 grafted molecules,residue 28 from the heavy chain was considered to be a non-criticalframework residue and the human residue (Thr28) was retained.Subsequently, however, it has been determined that residue 28 can beconsidered part of the CDR. Therefore, residue 28 was converted to thecorresponding mouse residue at that position (Ala28) using site directedmutagenesis with the oligonucleotide 5¹-GCT ACT GAA GGC GAA TCC AGA G-3′(SEQ ID NO:29). This change was later determined to not provide benefitover the human framework threonine at this site, and the threonine wasretained. The final grafted LM609 clone was designated huLM609M13 1135-4and is termed herein Vitaxin. The DNA sequence of clone Vitaxin is shownin FIGS. 2A and 2B.

EXAMPLE III Functional Characterization of Vitaxin

[0138] This Example shows the characterization of Vitaxin's bindingspecificity, affinity and functional activity in a number of in vitrobinding and cell adhesion assays.

[0139] The binding specificity of Vitaxin for the integrin α_(v)β₃ wasinitially assessed by measuring binding to α_(v)β₃ and itscrossreactivity to other α_(v)- or β₃-containing integrins.Specifically, binding specificity was assessed by measuring binding toα_(IIb)β₃, the major integrin expressed on platelets, and to α_(v)β₅, anintegrin found prevalent on endothelial cells and connective tissue celltypes.

[0140] Briefly, to determine crossreactivity, integrins were coated ontoan ELISA plate and a series of antibody dilutions were measured forVitaxin binding activity against α_(v)β₃ and the other integrins. Theintegrins α_(v)β₃ and α_(v)β₅ were isolated by affinity chromatographyas described by Cheresh (1987), supra, and Cheresh and Spiro (1987),supra. α_(IIb)β₃ was purchased from CalBiochem. Briefly, an LM609affinity column (Cheresh and Spiro (1987), supra) was used to isolateα_(v)β₃ from an octylglucoside human placental lysate, whereas ananti-α_(v) affinity column was used to isolate α_(v)β₅ from theα_(v)β₃-depleted column flow through. Antibody binding activity wasassessed by ELISA using a goat anti-human IgG-alkaline phosphataseconjugate. As a control, a purified human IgG₁ antibody was used sinceVitaxin contains a human IgG₁ backbone.

[0141] The results of this assay are shown in FIG. 4A and reveal thatVitaxin specifically binds to α_(v)β₃ with high affinity. There was nodetectable binding to the other α_(v)- or β₃-containing integrins atantibody concentrations over 1.0 mg/ml.

[0142] In a further series of binding studies, the binding affinity andspecificity was assessed in a competitive binding assay with theparental LM609 antibody against α_(v)β₃. Competitive binding wasmeasured in an ELISA assay as described above with LM609 being thelabeled antibody. Binding of LM609 was determined in the presence ofincreasing concentrations of Vitaxin competitor. Alternatively, thecontrol competitor antibody was again a human IgG₁.

[0143] The results of this competition are presented in FIG. 4B and showthat specific inhibition of LM609 binding can be observed at Vitaxinconcentrations of over 0.1 μg/ml. Almost complete inhibition is observedat Vitaxin concentrations greater than 100 μg/ml. This level ofcompetitive inhibition indicates that the parental monoclonal antibodyLM609 and the grafted version Vitaxin exhibit essentially identicalspecificity.

[0144] Binding affinity and specificity were also assessed by measuringthe inhibitory activity of Vitaxin on α_(v)β₃ binding to fibrinogen. Forthese studies, α_(v)β₃ was plated onto ELISA plates as described abovefor the Vitaxin/α_(v)β₃ binding studies. Inhibitory activity of Vitaxinwas determined by measuring the amount of bound biotinylated fibrinogenin the presence of increasing concentrations of Vitaxin or controlantibody. Briefly, fibrinogen was purchased from CalBiochem andbiotinylated with N-hydroxysuccinimidobiotin as described by themanufacturer (Pierce Life Science and Analytical Research). Streptavidinalkaline phosphatase was used to detect the bound fibrinogen.

[0145] The results of this assay are presented in FIG. 4C and reveal aspecific binding inhibition at Vitaxin concentrations higher than about0.1 μg/ml. These results, combined with those presented above showingspecific binding of Vitaxin to α_(v)β₃ and competitive inhibition ofLM609, demonstrate that Vitaxin maintains essentially all of the bindingcharacteristics and specificity exhibited by the parental murinemonoclonal antibody LM609. Described below are additional functionalstudies which corroborate these conclusions based on in vitro bindingassays.

[0146] Additional functional studies were performed to further assessthe specificity of Vitaxin binding. These studies were directed to theinhibition of integrin α_(v)β₃ binding in cell adhesion assays.Endothelial cell adhesion events are an important component in theangiogenic process and inhibition of α_(v)β₃ is known to reduce theneovascularization of tumors and thereby reduce the rate of tumorgrowth. The inhibition of α_(v)β₃-mediated cell attachment by Vitaxin inthese assays is indicative of the inhibitory activity expected when thisantibody is used in situ or in vivo.

[0147] Briefly, α_(v)β₃-positive M21 melanoma cells grown in RPMIcontaining 10% FBS were used for these cell binding assays. Cells werereleased from the culture dish by trypsinization and re-suspended inadhesion buffer at a concentration of 4×10⁵ cells/ml (see below)Vitaxin, LM609 or purified human IgG₁ (control antibody), were dilutedto the desired concentration in 250 μl adhesion buffer (10 mM Hepes, 2mM MgCl₂, 2 mM CaCl₂, 0.2 mM MnCl₂, and 1% BSA in Hepes buffered salineat pH 7.4) and added to wells of a 48-well plate precoated withfibrinogen. The fibrinogen was isolated as described above. Each wellwas coated with 200 μl fibrinogen at a concentration of 10 μg/ml for 1hour at 37° C. For the assay, an equal volume of cells (250 μl)containing Vitaxin, LM609 or isotype matched control antibody was addedto each of the wells, mixed by gentle shaking and incubated for 20minutes at 37° C. Unbound cells were removed by washing with adhesionbuffer until no cells remained in control wells coated with BSA alone.Bound cells were visualized by staining with crystal violet which wassubsequently extracted with 100 μl acetic acid (10%) and quantitated bydetermining the absorbance of the solubilized dye at 560 nm.

[0148] The results of this assay are shown in FIG. 5A and reveal thatboth Vitaxin and parental antibody LM609 inhibit M21 cell adhesion tofibrinogen over the same concentration range. The inhibitoryconcentration for 50% maximal adhesion was calculated to be about 50ng/ml. Specificity of Vitaxin was shown by the lack of inhibitionobserved by the control IgG₁ antibody.

[0149] In addition to the above cell adhesion results, the inhibitoryactivity of Vitaxin was also tested in an endothelial cell migrationassay. In this regard, the transwell cell migration assay was used toassess the ability of Vitaxin to inhibit endothelial cell migration(Choi et al., J. Vascular Surg., 19:125-134 (1994) and Leavesly et al.,J. Cell Biol, 121:163-170 (1993)).

[0150] Briefly, human umbilical vein endothelial cells in log phase andat low passage number were harvested by gentle trypsinization, washedand resuspended at a concentration of 2×10⁶ cells/ml in 37° C. HBScontaining 1% BSA (20 mM HEPES, 150 mM NaCl, 1.8 mM CaCl₂, 1.8 mM MgCl₂,5 mM KCl, and 5 mM glucose, pH 7.4). Antibodies (Vitaxin, LM609, andIgG₁ control) were diluted to 10 μg/ml from stock solutions. Antibodieswere added to cells in a 1:1 dilution (final concentration ofantibodies=5 μg/ml; final concentration of cells=1×10⁶ cells/ml) andincubated on ice for 10-30 minutes. The cell/antibody suspensions (200μl to each compartment) were then added to the upper compartments of aTranswell cell culture chamber (Corning Costar), the lower compartmentsof which had been coated with 0.5 ml of 10 μg/ml vitronectin (in HBS).Vitronectin serves as the chemoattractant for the endothelial cells. Thechambers were placed at 37° C. for 4 hours to allow cell migration tooccur.

[0151] Visualization of cell migration was performed by first removingthe remaining cells in the upper compartment with a cotton swab. Cellsthat had migrated to the lower side of the insert were stained withcrystal violet for 30 minutes, followed by solubilization in acetic acidand the absorbance of the dye was measured at a wavelength of 550 nm.The amount of absorbance is directly proportional to the number of cellsthat have migrated from the upper to the lower chamber. The results ofthe assay are presented in FIG. 7B. Both Vitaxin and the parentalantibody LM609 yielded essentially identical inhibitory results.Specifically, Vitaxin and LM609 inhibited about 60% of thevitronectin-induced migration of endothelial cells compared to the IgG₁control and to a sample with no inhibitor.

EXAMPLE IV Vitaxin-Mediated Inhibition of α_(v)β₃ In Animal Models

[0152] This Example describes the inhibition of tumor growth by Vitaxinin two animal models. Tumor growth was inhibited by inhibiting at leastα_(v)β₃-mediated neovascularization with Vitaxin.

[0153] The first model measures angiogenesis in the chickchorioallantoic membrane (CAM). This assay is a well recognized modelfor in vivo angiogenesis because the neovascularization of whole tissueis occurring. Specifically, the assay measures growth factor inducedangiogenesis of chicken CAM vessels growing toward the growthfactor-impregnated filter disk or into the tissue grown on the CAM.Inhibition of neovascularization is based on the amount and extent ofnew vessel growth or on the growth inhibition of tissue on the CAM. Theassay has been described in detail by others and has been used tomeasure neovascularization as well as the neovascularization of tumortissue (Ausprunk et al., Am. J. Pathol., 79:597-618 (1975); Ossonski etal. Cancer Res., 40:2300-2309 (1980); Brooks et al. Science, 264:569-571(1994a) and Brooks et al. Cell, 79:1157-1164 (1994b).

[0154] Briefly, for growth factor induced angiogenesis filter disks arepunched from #1 Whatman Qualitative Circles using a skin biopsy punch.Disks are first sterilized by exposure to UV light and then saturatedwith varying concentrations of TNF-α or HBSS as a negative control (forat least 1 hour) under sterile conditions. Angiogenesis is induced byplacing the saturated filter disks on the CAMs.

[0155] Inhibition of angiogenesis is performed by treating the embryoswith various amounts of Vitaxin and controls (antibody or purified humanIgG₁) . The treatments are performed by intravenous injectionapproximately 24 hours after disk placement. After 48 hours, CAMs aredissected and angiogenesis is scored on a scale of 1-4. HBSS saturatedfilter disks are used as the negative control, representing angiogenesisthat may occur in response to tissue injury in preparing CAMs, and,values for these CAMS are subtracted out as background. Purified humanIgG₁ is used as the negative control for injections since Vitaxin is ofthe human IgG₁ subclass. Vitaxin was found to inhibit TNF-α inducedangiogenesis in a dose dependent manner. Maximal inhibition occurredwith a single dose of Vitaxin at 300 μg which resulted in greater than80% inhibition compared to the human IgG₁ control.

[0156] In addition to the above described CAM assay using growthfactor-induced neovascularization, additional studies were performedutilizing tumor-induced neovascularization. For these assays,angiogenesis was induced by transplantating of α_(v)β₃-negative tumorfragments into the CAMs. The use of α_(v)β₃-negative tumor fragmentsensures that any inhibition of tumor growth is due to the inhibition ofα_(v)β₃-mediated neovascularization by CAM-derived endothelial cells andnot to adhesion events mediated by α_(v)β₃ present on the tumor cells.

[0157] Inhibition of tumor growth was assessed by placing a single cellsuspension of FG (8×10⁶ cells, pancreatic carcinoma) and HEp-3 cells(5×10⁵ cells, laryngeal carcinoma) onto CAMs in 30 μl. One week later,tumors are removed and cut into approximately 50 mg fragments at whichtime they are placed onto new CAMs. After 24 hours of this secondplacement embryos are injected intravenously with Vitaxin or human IgG₁as a negative control. The tumors are allowed to grow for about 7 daysfollowing which they are removed and weighed.

[0158] The results of Vitaxin treatment on the neovascularization oftumors is shown in FIG. 6A. The data is expressed as a mean change intumor weight and demonstrate that Vitaxin is able to inhibit the growthof α_(v)β₃-negative tumors such as FG and HEp-3 tumor fragments. Morespecifically, there was a mean weight change for Vitaxin treated FGtumor fragments of −5.38 whereas a change of −11.0 was observed forVitaxin treated HEp-3 tumors. The IgG₁ controls exhibited positive meanweight changes of 25.29 and 28.5 for the FG and HEp-3 tumor fragments,respectively. These results were obtained following a single intravenousinjection.

[0159] In a second animal model, the inhibition of Vx2 carcinoma cellsin rabbits was used as a measure of Vitaxin's inhibitory effect ontumors. The Vx2 carcinoma is a transplantable carcinoma derived from aShope virus-induced papilloma. It was first described in 1940 and hassince been used extensively in studies on tumor invasion, tumor-hostinteractions and angiogenesis. The Vx2 carcinoma is fibrotic in nature,highly aggressive, and exhibits features of an anaplastic typecarcinoma. Propagation of Vx2 tumor is accomplished through serialtransplantation in donor rabbits. Following subcutaneoustransplantation, it has been reported that after an initial inflammatoryreaction, host repair mechanisms set in between days 2 and 4. Thisrepair mechanism is characterized by the formation of new connectivetissue and the production of new capillaries. The newly formedcapillaries are restricted to the repair zone at day 4, however, by day8 they have extended to the outer region of the tumor. Thesecharacteristics and the pharmacokinetics of Vitaxin in rabbits were usedto determine initial doses and scheduling of treatments for theseexperiments. The elimination half life of Vitaxin in animal serum dosedat 1, 5, and 10 mg/kg was found to be 38.9, 60.3, and 52.1 hours,respectively.

[0160] Growth of Vx2 tumors in the above animal model was used to studythe effect of Vitaxin after early administration on primary tumor growthin rabbits implanted subcutaneously with Vx2 carcinoma. Briefly, Vx2tumors (50 mg) were transplanted into the inner thigh of rabbits throughan incision between the skin and muscle. Measurements of the primarytumor were taken throughout the experiment through day 25. At day 28after the transplantation animals were sacrificed and tumors wereexcised and weighed. By day 28, tumors became extremely irregular inshape and as a result, measurements became difficult and were notreflective of tumor volume. Therefore measurements were assessed onlythrough day 25.

[0161] In a first study, rabbits were treated starting at day 1 posttumor implantation with 5 and 1 mg/kg Vitaxin every four days for 28days for a total of 7 doses). In both groups, inhibition of tumor growthwas observed. In a second series of studies, rabbits were treatedbeginning at day 7 post tumor implantation as described above for atotal of 5 doses. Inhibition of tumor growth was also observed.

[0162] It should be noted that administering a grafted antibody as arepeat dose treatment to rabbits might generate an immune response thatcan have a neutralizing effect on Vitaxin thus potentially comprisingefficacy. Preliminary data suggest that approximately 25-50% of theanimals develop such a response.

[0163] The results of each of the Vitaxin treatments described above isshown in FIG. 6B and 6C. In the rabbits receiving treatments on day 1,inhibition of tumor growth was observed in both the 1 mg/kg and the 5mg/kg dosing groups compared to the control PBS treated control.Specifically, a growth inhibition of about 67 and 80% was observed,respectively, as measured by the mean tumor weight. A lesser degree ofinhibition was observed in animals that began Vitaxin treatment on day 7post implantation. These results are shown in FIG. 6C. In all cases,inhibition of tumor growth was not see at Vitaxin concentrations lowerthan 0.2 mg/kg.

EXAMPLE V Construction of LM609 Grafted Functional Antibody Fragments

[0164] This Example shows the construction of functional LM609 graftedantibody fragments in which only the CDRs have been transferred from theLM609 donor antibody to a human acceptor framework.

[0165] CDR grafting of LM609 to produce a functional antibody fragmentwas accomplished by the methods set forth below. These procedures areapplicable for the CDR grafting of essentially any donor antibody whereamino acid residues outside of the CDRs from the donor antibody are notdesired in the final grafted product.

[0166] Briefly, the protein sequence of the LM609 antibody, wasdetermined by cloning and sequencing the cDNA that encodes the variableregions of the heavy and light chains as described in Example I. TheCDRs from the LM609 donor antibody were identified and grafted intohomologous human variable regions of a human acceptor framework.Identification of CDR regions were based on the combination ofdefinitions published by Kabat et al., and MacCallum et al.

[0167] The boundaries of the CDR regions have been cumulatively definedby the above two publications and are residues 30-35, 47-66 and 97-106for CDRs 1, 2 and 3, respectively, of the heavy chain variable regionand residues 24-36, 46-56, and 89-97 for CDRs 1, 2 and 3, respectively,of the light chain variable region. Non-identical donor residues withinthese boundaries but outside of CDRs as defined by Kabat et al. wereidentified and were not substituted into the acceptor framework.Instead, functional non-donor amino acid residues were identified andsubstituted for certain of these non-identical residues.

[0168] As described below, the only non-identical residue outside of theCDRs as defined by Kabat et al. but within the CDRs as defined above isat position 49 of the LM609 light chain. To identify functionalnon-donor amino acids at this position, a library of nineteen antibodieswas constructed that contained all non-donor amino acids at position 49and then screened for binding activity against α_(v)β₃.

[0169] Human immunoglobulin sequences were identified from theBrookhaven Protein Data Bank-Kabat Sequences of Proteins ofImmunological Interest database (release 5.0). Human framework sequencesshowing significant identity to the murine LM609 variable region genesequences were selected for receiving the LM609 CDRs. Human heavy chainvariable region M72 ′CL had 88% identity to frameworks 1, 2 and 3 ofLM609 heavy chain and human light chain V region LS1 ′CL had 79%identity to frameworks 1, 2 and 3 of LM609 light chain. With theexclusion of non-identical residues outside of the CDRs as defined byKabat et al. murine LM609 CDR sequences as defined by Kabat et al. andMacCallum et al. were grafted onto the human frameworks. Using thisgrafting scheme, the final grafted product does not contain any aminoacid residues outside of the CDRs as defined by Kabat et al. which areidentical to an LM609 amino acid at the corresponding position (outsideof residues: 31-35, 50-66 and 99-106 for CDRs 1, 2 and 3, respectively,of the heavy chain variable region and residues 24-34, 50-56, and 89-97for CDRs 1, 2 and 3, respectively, of the light chain variable region).Moreover, no intermediates are produced which contain an amino acidresidue outside of the CDRs as defined by Kabat et al. which areidentical to the LM609 amino acid at that position. The CDR graftingprocedures are set forth below.

[0170] Full-length CDR grafted variable region genes were synthesized byPCR using long overlapping oligonucleotides as described previously inExample II. The heavy chain variable region oligonucleotides were thosedescribed previously as SEQ ID NOS:9-13. The light chain variable regionoligonucleotides were synthesized so as to contain the CDR graftedvariable region as well as a stop codon at position 49. The fiveoligonucleotides for the light chain LM609 grafted variable region areshow as SEQ ID NOS:23-27 where the second oligonucleotide in the seriescontains the stop codon at position 49 (SEQ ID NO:24). The nucleotidesequences of oligonucleotides used to construct LM609 grafted lightchain variable region is shown in Table 7. TABLE 7 Oligonucleotides Usedto Construct LM609 Grafted Light Chain Variable Region GAGATTGTGCTAACTCAGTC TCCAGCCACC CTGTCTCTCA GCCCAGGAGA AAGGGCGACT SEQ ID NO:23CTTTCCTGCC AGGCCAGCCA AAGTATT TTAGATGAGA AGCCTTGGGG CTTGACCAGGCCTTTGTTGA TACCAGTGTA GGTGGTTGCT SEQ ID NO:24 AATACTTTGG CTGGCCCAAGGCTTC TCATCTAATA TCGTTCCCAG TCCATCTCTG GGATCCCCGC CAGGTTCAGT SEQ IDNO:25 GGCAGTGGAT CAGGGACAGA TTTC GCTGCCACTC TGTTGACAGT AATAGACTGCAAAATCTTCA GGCTCCAGAC TGGAGATAGT SEQ ID NO:26 GAGGGTGAAA TCTGTCCCTG ACAACAGAGTG GCAGCTGGCC TCACACGTTC GGAGGGGGGA CCAAGGTGGA AATTAAG SEQ IDNO:27

[0171] All long oligonucleotides were gel purified. CDR grafting of theLM609 heavy chain variable region was constructed by mixing 5overlapping oligonucleotides (SEQ ID NOS:9-13), at equimolarconcentrations, in the presence of annealing PCR primers containing atleast 18 nucleotide residues complementary to vector sequences for theefficient annealing of the amplified V region product to thesingle-stranded vector. The annealed mixture was fully converted to adouble-stranded molecule with T4 DNA polymerase plus dNTPs and ligatedwith T4 ligase. The mutagenesis reaction (1 μl) was electroporated intoE. coli strain DH10B (BRL), titered onto a lawn of XL-1 (Stratagene,Inc.) and incubated until plaques formed. Replica filter lifts wereprepared and plaques containing V_(H) gene sequences were screenedeither by hybridization with a digoxigenin-labeled oligonucleotidecomplementary to LM609 heavy chain CDR 2 sequences or reactivity with7F11-alkaline phosphatase conjugate, a monoclonal antibody raisedagainst the decapeptide sequence Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser(SEQ ID NO:28) appended to the carboxy terminus of the vector CH₁ domain(Biosite, Inc., San Diego, Calif.).

[0172] Fifty clones that were double-positive were pooled and used toprepare uridinylated template for hybridization mutagenesis with theamplified CDR grafted LM609 V_(L) product constructed in a similarfashion using the five overlapping oligonucleotides shown as SEQ IDNOS:23-27. The mutagenesis reaction was electroporated into E. colistrain DH10B. Randomly picked clones were sequenced to identify aproperly constructed template for construction of the non-donor libraryat position 49. This template was prepared as a uridinylated templateand an oligonucleotide population of the following sequence was used forsite directed mutagenesis.

GGGAACGATA-19aa-GATGAGAAGC

[0173] The sequence 19aa in the above primer (SEQ ID NO:30) representsthe fact that this primer specifies a sequence population consisting of19 different codon sequences that encode each of the 19 non-donor aminoacids. These amino acids are those not found at position 49 of LM609 andinclude all amino acids except for Lys. Clones that resulted from thismutagenesis were picked and antibody expressed by these clones wereprepared. These samples were then screened for binding to αvβ₃ in anELISA assay. Clones having either Arg or Met amino acids in position 49were functionally identified. The nucleotide and amino acid sequence ofthe LM609 grafted heavy chain variable region is show in FIG. 1A (SEQ IDNOS:1 and 2, respectively). The nucleotide and amino acid sequence ofthe LM609 grafted light chain variable region is shown in FIG. 7 (SEQ IDNOS:31 and 32, respectively).

EXAMPLE VI Generation of LM609 Grafted Antibodies Having EnhancedActivity

[0174] This example shows in vitro maturation of LM609 grafted antibodyto obtain antibody variants having increased affinity to α_(v)β₃relative to the parent LM609 grafted antibody.

[0175] To optimize the affinity of LM609 grafted antibody in vitro, anM13 phage system was used, which permits the efficient synthesis,expression, and screening of libraries of functional antibody fragments(Fabs). The contribution of each of the six CDRs of the Ig heavy andlight chains was assessed. The CDRs were defined broadly based on acombination of sequence variability and antibody structural models(Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia et al.,supra; MacCallum et al., supra). Thus, one library was constructed foreach CDR, with the exception of H2 which was split into two librariesdue to its long (20 amino acids) length. The variable region frameworkswhich harbored the mutated CDRs were the heavy chain variable regionshown in FIG. 1a (SEQ ID NO:2) and the light chain variable region shownin FIG. 7 (SEQ ID NO:32).

[0176] CDRs were chosen from the heavy chain variable region shown inFIG. 1a (SEQ ID NO:2) and the light chain variable region shown in FIG.7 (SEQ ID NO:32). Briefly, utilizing the numbering system of Kabat etal., supra, the residues chosen for mutagenesis of the CDRs (Table 9)were: Gln²⁴-Tyr³⁶ in light chain CDR1 (L1); Leu⁴⁶-Ser⁵⁶ in light chainCDR2 (L2); Gln⁸⁹-Thr⁹⁷ in light chain CDR3 (L3); Gly²⁶-Ser³⁵ in heavychain CDR1 (H1); Trp⁴⁷-Gly⁶⁵ in heavy chain CDR2 (H2); and Ala⁹³-Tyr¹⁰²in heavy chain CDR3 (H3). Libraries were created for each CDR, with theoligonucleotides designed to mutate a single CDR residue in each clone.Due to the extended length of H2, two libraries mutating residues 47-55(H2a) and 56-65 (H2b), respectively, were constructed to cover thisregion.

[0177] The template for generating light chain CDR3 mutants containedGly at position 92. However, it was subsequently determined thatposition 92 of the light chain CDR3 was inadvertently deduced to be aGly, resulting in humanized LM609 grafted antibodies being constructedwith Gly at that position. It was later realized that the original LM609sequence contained an Asn at position 92. Using the methods describedherein to introduce mutations into CDRs of an LM609 grafted antibody, anLM609 grafted antibody having Asn at position 92 of light chain CDR3 wasfound to have α_(v)β₃ binding activity (see Table 9), confirming theidentification of Asn⁹² as a functional LM609 grafted antibody. Thus,antibodies containing light chain CDR3 having Gly or Asn at position 92are active in binding α_(v)β₃.

[0178] Oligonucleotides encoding a single mutation were synthesized byintroducing NN(G/T) at each CDR position as described previously (Glaseret al., supra). The antibody libraries were constructed in Ml31XL604vector by hybridization mutagenesis as described previously, with somemodifications (Rosok et al., J. Biol. Chem. 271:22611-22618 (1996); Huseet al., J. Immunol. 149:3914-3920 (1992); Kunkel, Proc. Natl. Acad. Sci.USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. 154:367-382(1987)). Briefly, the oligonucleotides were annealed at a 20:1 molarratio to uridinylated LM609 grafted antibody template (from which thecorresponding CDR had been deleted) by denaturing at 85° C. for 5 min,ramping to 55° C. for 1 h, holding at 55° C. for 5 min, then chilling onice. The reaction was extended by polymerization electroporated intoDH10B and titered onto a lawn of XL-1 Blue. The libraries consisted ofpools of variants, each clone containing a single amino acid alterationin one of the CDR positions. Utilizing codon-based mutagenesis, everyposition in all of the CDRs was mutated, one at a time, resulting in thesubsequent expression of all twenty amino acids at each CDR residue(Glaser et al., supra). The CDR libraries ranged in size from 288 (L3)to 416 (L1) unique members and contained a total of 2336 variants.

[0179] To permit the efficient screening of the initial libraries, ahighly sensitive plaque lift assay, termed capture lift, was employed(Watkins et al., Anal. Biochem. 256 (1998)). Briefly, phage expressionlibraries expressing LM609 grafted antibody variants were initiallyscreened by a modified plaque lift approach, in which the nitrocellulosewas pre-coated with goat anti-human kappa antibody and blocked withbovine serum albumin prior to application to the phage-infectedbacterial lawn. Following the capture of phage-expressed LM609 graftedantibody variant Fabs, filters were incubated with 1.0 μg/mlbiotinylated α_(v)β₃ for 3 h at 4° C., washed four times, incubated with2.3 μg/ml NeutrAvidin-alkaline phosphatase (Pierce Chemical Co.;Rockford, Ill.) for 15 min at 25° C., and washed four times. Alldilutions and washes were in binding buffer. Variants that bound α_(v)β₃were identified by incubating the filters for 10-15 min in 0.1M Tris, pH9.5, containing 0.4 mM 2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene)ditetrazolium chloride and 0.38 mM5-bromo-4-chloro-3-indoxyl phosphate mono-(p-toluidinium) salt (JBLScientific, Inc.; San Luis Obispo, Calif.).

[0180] To generate biotinylated α_(v)β₃, the α_(v)β₃ receptor waspurified from human placenta by affinity chromatography, as describedpreviously (Smith and Cheresh, J. Biol. Chem. 263:18726-18731 (1988)).To biotinylate α_(v)β₃, purified receptor was dialyzed into 50 mM HEPES,pH 7.4, 150 mM NaCl, 1.0 mM CaCl₂₁ containing 0.1% NP-40 (bindingbuffer) and incubated with 100-fold molar excess sulfosuccinimidobiotinfor 3 h at 4° C. The reaction was terminated by the addition of 50 mMethanolamine.

[0181] Phage expressed LM609 grafted antibody variants were selectivelycaptured on nitrocellulose filters coated with goat anti-human kappachain antibody, probed with biotinylated α_(v)β₃, and detected withNeutrAvidin-alkaline phosphatase. Initially, biotinylated α_(v)β₃ wastitrated on lifts containing phage expressing the LM609 grafted antibodyparent molecule only. Subsequently, the concentration of biotinylatedα_(v)β₃ was decreased to yield a barely perceptible signal. In this way,only clones expressing higher affinity variants were readily identifiedduring screening of the variant libraries. Following the exhaustivecapture lift screening of ≧2500 clones from each library, 300 higheraffinity variants were identified (see Table 10). The greatest number ofclones displaying improved affinity were identified in the H3 (185) andL3 (52) CDRs, though variants with improved affinity were identified inevery CDR.

[0182] LM609 grafted antibody variants identified by capture lift ashaving α_(v)β₃ binding activity were further characterized to determinebinding affinity to α_(v)β₃ specificity for α_(v)β₃ over otherintegrins, and α_(v)β₃ association and dissociation rates. For theseassays, purified Fab of LM609 grafted antibody variants was used.Briefly, Fab was expressed as described previously and was released fromthe periplasmic space by sonic oscillation (Watkins et al., supra,1997). Cells collected from one liter cultures were lysed in 10 ml 50 mMTris, pH 8.0, containing 0.05% Tween 20. Fab was bound to a 1 ml proteinA column (Pharmacia) which had been equilibrated with 50 mM glycine, pH8, containing 250 mM NaCl, washed with the same buffer, and eluted with10 ml of 100 mM glycine, pH 3, into one-tenth volume 1M Tris, pH 8.Purified Fab was quantitated as described previously (Watkins et al.,supra, 1997).

[0183] LM609 grafted antibody variants were tested for binding toα_(v)β₃ and specificity of binding to α_(v)β₃ relative to α_(v)β₅ andα_(IIb)β₃. For ELISA titration of Fab on immobilized α_(v)β₃ and therelated integrins α_(v)β₅ and α_(IIb)β₃, Immulon II microtiter plateswere coated with 1 μg/ml purified receptor in 20 mM Tris, pH 7.4, 150 mMNaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, washed once, and blocked in 3%BSA in 50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂ for 1 hat 25° C. Human α_(IIb)β₃, purified from platelets, was obtained fromEnzyme Research Laboratories, Inc. (South Bend, Ind.) and α_(v)β₅ waspurified from placental extract depleted of α_(v)β₃, as describedpreviously (Smith et al., J. Biol. Chem. 265:11008-11013 (1990)). Justprior to use, the plates were washed two times and were then incubated 1h at 25° C. with various dilutions of Fab. The plates were washed fivetimes, incubated 1 h at 25° C. with goat anti-human kappa-alkalinephosphatase diluted 2000-fold, washed five times, and developed asdescribed previously (Watkins et al., supra, 1997). All dilutions andwashes were in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl₂, and 1 mMMgCl₂. TABLE 10 Capture Lift Screenincr of LM609 grafted antibody CDRLibraries. Enhanced Library Size¹ Screened² Positives³ Affinity⁴ H1 3202500 16  8 H2a 320 5000 26  7 H2b 320 5000 2  1 H3 320 5000 185 78⁵ L1416 2500 12  1 L2 352 3250 7  1 L3 288 5000 52 41

[0184]FIG. 8 shows titration of antibody variants and LM609 graftedantibody Fab on immobilized α_(v)β₃. Bacterial cell lysates containingLM609 grafted antibody (closed circles), variants with improved affinityisolated from the primary libraries (S102, closed squares; Y100, opensquares; and Y101, open triangles) or from the combinatorial libraries(closed triangles), or an irrelevant Fab (open circles) were titrated onimmobilized α_(v)β₃.

[0185] Comparison of the inflection points of the binding profilesobtained from titrating variants on immobilized α_(v)β₃ demonstratedthat multiple clones displayed >3-fold improved affinity, confirming theeffectiveness of utilizing the capture lift in a semi-quantitativefashion (FIG. 8, compare squares and open triangles with closedcircles). Based on the capture lift screening and subsequentcharacterization of binding to immobilized α_(v)β₃ it was concluded thatboth heavy and light chain CDRs are directly involved in the interactionof α_(v)β₃ with the LM609 grafted antibody variants.

[0186] DNA was isolated from clones displaying >3-fold enhanced bindingand sequenced to identify the mutations which resulted in higheraffinity. DNA sequencing was performed on isolated single-stranded DNA.The heavy and light chain variable region genes were sequenced by thefluorescent dideoxynucleotide termination method (Perkin-Elmer; FosterCity, Calif.). Based on sequence analysis of 103 variants, 23 uniquemutations clustered at 14 sites were identified (Table 9). The majorityof the sites of beneficial mutations were found in the heavy chain CDRs,with four located in H3, and three each in H2 (2a and 2b combined) andH1. Seven distinct and beneficial amino acid substitutions wereidentified at a single site within H3, tyrosine residue 102. The diversenature of the substitutions at this site suggests that tyrosine residue102 may sterically hinder LM609 grafted antibody binding to α_(v)β₃. Insupport of this, variants expressing the other aromatic amino acids(phenylalanine, histidine, and tryptophan) instead of tyrosine atresidue 102 were never isolated following screening for enhancedbinding.

[0187] The affinities of select variants were further characterized byutilizing surface plasmon resonance (BIAcore) to measure the associationand dissociation rates of purified Fab with immobilized α_(v)β₃ Briefly,surface plasmon resonance (BIAcore; Pharmacia) was used to determine thekinetic constants for the interaction between α_(v)β₃ and LM609 graftedantibody variants. Purified α_(v)β₃ receptor was immobilized to a(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride)/N-hydroxysuccinimide-activated sensor chip by injecting30 μl of 15 μg/ml α_(v)β₃ in 10 mM sodium acetate, pH 4. To obtainassociation rate constants (k_(on)), the binding rate at five differentFab concentrations, ranging from 5-40 μg/ml in 50 mM Tris-HCl, pH 7.4,100 mM NaCl, 2 mM CaCl₂, and 1 mM MgCl₂, was determined at a flow rateof 10 μl/min. Dissociation rate constants (k_(off)) were the average offive measurements obtained by analyzing the dissociation phase at anincreased flow rate (40 μl/min). Sensorgrams were analyzed with theBIAevaluation 2.1 program (Pharmacia). Residual Fab was removed aftereach measurement with 10 mm HCl, 2 mM CaCl₂ and 1 mM MgCl₂.

[0188] Table 9 shows that the variants all displayed a lower Kd than theLM609 grafted antibody parent molecule, consistent with both the capturelift and the ELlSA. Analysis of association and dissociation ratesrevealed that the majority of improved variants had slower dissociationrates while having similar association rates. For example, LM609 graftedantibody had an association rate 18.0×10⁴ M⁻¹s⁻¹, while the variantsranged from 16.7-31.8×10⁴ M⁻¹s⁻¹. In contrast, every clone dissociatedslower than LM609 grafted antibody (4.97×10⁻³ S⁻¹) with dissociationrates ranging from 1.6-fold (3.03×10⁻³ s⁻¹) to 11.8-fold (0.42×10⁻³ s⁻¹)slower.

[0189] These results demonstrate that introducing single amino acidsubstitutions into LM609 grafted antibody CDRs allows the identificationof modified LM609 grafted antibodies having higher affinity for α_(v)β₃than the parent LM609 grafted antibody. TABLE 9 Identification ofEnhanced LM609 Grafted Antibodies from Primary Libraries k_(on) (× 10⁴)k_(off) (× 10⁻³) chain* library† sequence (M⁻¹s⁻¹) (s⁻¹) Kd (nM) LM60918.0 4.97 27.6 grafted antibody H CDR1 G F T F S S Y D M S T27   T n.d.n.d. n.d. W29       W n.d. n.d. n.d. L30         L n.d. n.d. n.d. HCDR2a   W V A K V S S G G G K52             K 17.8 2.18 12.2 H CDR2b  S T Y Y L D T V Q G P60           P 31.8 1.85 5.8 E64                  E n.d. n.d. n.d. H CDR3   A R H N Y G S F A Y H97          H 22.0 3.03 13.8 Y100                 Y 17.5 2.51 14.3 D101                  D n.d. n.d. n.d. Y101                   Y 21.8 0.482.2 S102                     S 24.2 1.44 6.0 T102                     T24.6 1.43 5.8 D102                     D 27.6 0.97 3.5 E102                    E n.d. n.d. n.d. M102                     M n.d.n.d. n.d. G102                     G 16.1 2.01 12.5 A102                    A 27.5 2.27 8.3 L CDR1 Q A S Q S I S N H L H W Y F32                F 16.7 0.42 2.5 L CDR2     L L I R Y R S Q S I S S51              S n.d. n.d. n.d. L CDR3         Q Q S G S W P H T N92              N 23.6 1.35 5.7 T92               T n.d. n.d. n.d. L96                      L 24.3 2.23 9.2 Q96                       Q n.d.n.d. n.d.

EXAMPLE VII Generation of High Affinity LM609 Grafted Antibodies

[0190] This example shows that single amino acid mutations in CDRs of anLM609 grafted that result in higher affinity binding to α_(v)β₃ can becombined to generate high affinity LM609 grafted antibodies.

[0191] Random combination of all of the beneficial mutations of LM609grafted antibody would generate a combinatorial library containing >10⁵variants, requiring efficient screening methodologies. Therefore, todetermine if clones displaying >10-fold enhanced affinities could berapidly distinguished from one another, variants displaying 3 to 13-foldenhanced affinity were evaluated by capture lift utilizing lowerconcentrations of biotinylated α_(v)β₃ Despite repeated attempts with abroad range of concentrations of α_(v)β₃, consistent differences in thecapture lift signals were not observed. Because of this, smallercombinatorial libraries were constructed and subsequently screened byELlSA.

[0192] Four distinct combinatorial libraries were constructed in orderto evaluate the optimal number of combinations that could beaccomplished utilizing two site hybridization mutagenesis (FIG. 9).Briefly, combinatorial libraries were constructed by synthesizingdegenerate oligonucleotides encoding both the wild-type and beneficialheavy chain mutations (H2, Leu⁶⁰→Pro; H3 Tyr⁹⁷→His; H3, Ala¹⁰¹→-Tyr; H3,Tyr¹⁰²→Ser, Thr, Asp, Glu, Met, Gly, Ala). Utilizing two sitehybridization mutagenesis, as described above, the oligonucleotides wereannealed at a 40:1 molar ratio to uridinylated template prepared fromLM609 grafted antibody and three light chain mutations (FIG. 9; L1,His³²→Phe; L3, Gly⁹²→Asn; L3, His⁹⁶→Leu). As a result, a total of 256variants were synthesized in four combinatorial library subsets.

[0193]FIG. 9 shows construction of combinatorial libraries of beneficialmutations. Uridinylated template from LM609 grafted antibody and threeoptimal light chain variants (F32, N92, and L96) was prepared. Two sitehybridization was performed with two degenerate oligonucleotides, whichwere designed to introduce beneficial mutations at four distinct heavychain residues.

[0194] Following preparation of uridinylated templates of LM609 graftedantibody and three light chain variants, (Table 9; F32, N92, and L96),degenerate oligonucleotides encoding the wild type residue and the mostbeneficial heavy chain mutations (Table 9; P60, H97, Y101, S102, T102,D102, E102, M102, G102, and A102) were hybridized to the light chaintemplates, resulting in four combinatorial libraries, each containing 64unique variants. Potentially, the combination of multiple mutations canhave detrimental effects on affinity and, thus, can prevent theidentification of beneficial combinations resulting from mutations atfewer sites. For this reason, the amino acid expressed by the LM609grafted antibody parent molecule was included at each position in thecombinatorial library. By utilizing this approach, simultaneouscombinatorial mutagenesis of three CDRs (L1 or L3 each in combinationwith H2 and H3) was accomplished. Based on sequence analysis, the twosite hybridization mutagenesis was achieved with ˜50% efficiency.

[0195] In order to screen the combinatorial libraries, soluble Fab wasexpressed and released from the periplasm of small-scale (<1 ml)bacterial cultures that had been infected with randomly selected clones.Although variable expression levels were observed, uniform quantities ofthe unpurified variants were captured on a microtiter plate through apeptide tag present on the carboxyl-terminus of the heavy chain.Briefly, combinatorial LM609 grafted antibody libraries were screened byan ELlSA that permits the determination of relative affinities ofantibody variants produced in small-scale bacterial cultures (Watkins etal., Anal. Biochem. 253:37-45 (1997)). An Immulon II microtiter plate(Dynatech Laboratories; Chantilly, Va.) was coated with 10 μg/ml of the7F11 monoclonal antibody, which recognizes a peptide tag on thecarboxyl-terminus of the LM609 grafted antibody variant heavy chains(Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)). Following captureof Fab from E. coli lysates, the plate was incubated with 0.5-1 μg/mlbiotinylated α_(v)β₃ for 1 h at 25° C. The plate was washed seven times,incubated with 0.5 U/ml streptavidin-alkaline phosphatase (1000 U/ml;Boehringer Mannheim; Indianapolis, Ind.) for 15 min at 25° C., washedseven times, and developed as described previously (Watkins et al.,supra, 1997). All dilutions and washes were in binding buffer.

[0196] As described previously (Watkins et al., supra, 1997), this ELISAscreening method enabled a rapid and direct comparison of the relativeaffinities of the variants following incubation with biotinylatedα_(v)β₃ and streptavidin-alkaline phosphatase. To ensure that the fullFab diversity was sampled, one thousand randomly selected clones werescreened from each combinatorial library. Variants that displayed anenhanced ELlSA signal were further characterized for binding toimmobilized α_(v)β₃ (FIG. 8, closed triangles) and were sequenced toidentify the mutations (Table 10).

[0197] Screening of the four combinatorial libraries identified fourteenunique combinations of mutations that improved binding significantlyover the individual mutations identified in the screening of the firstlibrary. While the best clone from the primary screen had a 12.5-foldincrease in affinity, the fourteen unique combinations isolated fromscreening the combinatorial libraries displayed affinities ranging from18 to 92-fold greater than the parent LM609 grafted antibody. Themajority of these variants consisted of H2 and H3 mutations combinedwith the L1 or L3 mutations. Beneficial combinations of heavy chainmutations with wild-type light chain were also identified, but did notresult in improved affinity to the same extent as other combinatorialvariants. The variants predominantly contained 2 to 4 mutations, withone clone, C29, containing five mutations. No direct correlation betweenthe total number of mutations in each variant and the resulting affinitywas observed. For example, while the binding of clone C37 was 92-foldenhanced over the parent molecule and was achieved through thecombination of three mutations, clone C29 had ˜55-fold greater affinityachieved through the combination of five mutations. Multiple variantsdisplaying >50-fold enhanced affinity resulting from the combination ofas few as two mutations were identified (2G4, 17, and V357D).

[0198] The combinatorial clones with improved affinity alldisplayed >10-fold slower dissociation rates, possibly reflecting aselection bias introduced by long incubation steps in the screening. Inaddition, all of the combinatorial variants isolated from the librarybased on the L96 light chain mutation also displayed 2 to 4-fold greaterassociation rates. Previously, it has been demonstrated that theantibody repertoire shifts towards immunoglobulins displaying higherassociation rates during affinity maturation in vivo (Foote andMilstein, Nature 352:530-532 (1991)). The L96 subset of variants,therefore, may more closely mimick the in vivo affinity maturationprocess where B-lymphocyte proliferation is subject to a kineticselection.

[0199] LM609 grafted antibody binds the α_(v)β₃ complex specifically anddoes not recognize either the α_(v) or the β₃ chain separately. Tofurther characterize the variants, clones were screened for reactivitywith the related integrins, α_(IIb)β₃ and α_(v)β₅. All variants testedwere unreactive with both α_(IIb)β₃ and α_(v)β₅, consistent with theimproved binding not substantially altering the interaction of Fab andreceptor. TABLE 10 Identification of Optimal Combinatorial Mutationssequence† L1 L3 L3 H2 H3 H3 H3 k_(on) (× 10⁴) k_(on) (× 10⁻³) library*clone 32 92 96 60 97 101 102 (M⁻¹s⁻¹) (s⁻¹) Kd (nM) wild type H G H L YA Y 18.0 4.97 27.6 F32 17 F S 25.1 0.138 0.5 7 F P H S 20.4 0.236 1.2 56F P S 26.6 0.135 0.5 C59 F P D 26.5 0.137 0.5 C176 F P T 22.5 0.192 0.9V357D F D 27.9 0.140 0.5 N92 C119 N P S 21.5 0.316 1.5 L96 8F9 L P H S47.5 0.280 0.6 C29 L P H Y S 67.5 0.343 0.5 2G4 L S 60.3 0.229 0.4 6H6 LH S 50.4 0.187 0.4 C37 L Y E 44.8 0.147 0.3 6D1 L P Y S 41.0 0.158 0.46G1 L P S 38.9 0.280 0.7

[0200] As a first step toward determining if the increase in affinity ofthe variants resulted in greater biological activity, variantsdisplaying a range of affinities were assayed for their ability toinhibit the binding of a natural ligand, fibrinogen, to immobilizedα_(v)β₃ receptor. Briefly, LM609 grafted antibody variants were testedfor inhibition of ligand binding as described previously except that thebinding of biotinylated human fibrinogen (Calbiochem, La Jolla, Calif.)was detected with 0.5 μg/ml NeutrAvidin-alkaline phosphatase (Smith etal., J. Biol. Chem. 265:12267-12271 (1990)).

[0201] The results of these competition assays are shown in FIG. 10.FIG. 10A shows inhibition of fibrinogen binding to immobilized α_(v)β₃.Immobilized α_(v)β₃ was incubated with 0.1 μg/ml biotinylated fibrinogenand various concentrations of LM609 grafted antibody (open circles),S102 (closed circles), F32 (open triangles), or C59 (closed triangles)for 3 h at 37° C. Unbound ligand and Fab were removed by washing andbound fibrinogen was quantitated following incubation with NeutrAvidinalkaline phosphatase conjugate. FIG. 10B shows correlation of affinityof variants with inhibition of fibrinogen binding. The concentration ofvariants required to inhibit the binding of fibrinogen to immobilizedα_(v)β₃ by 50% (IC₅₀) was plotted as a function of the affinity (Kd).

[0202] As shown in FIG. 10A, higher affinity variants were moreeffective at blocking the ligand binding site of the receptor (compareLM609 grafted antibody, open circles, with any of the variants).Subsequent analysis of ten variants displaying affinities (Kd) rangingfrom 0.3 to 27 nm demonstrated a good correlation (r²=0.976) betweenaffinity and ability to inhibit fibrinogen binding (FIG. 10B). Inaddition, the variants were tested for inhibition of vitronectin bindingto the receptor. Similar to fibrinogen, the variants were more effectiveat inhibiting the interaction than the parent molecule. Thus, consistentwith the cross-reactivity studies with related integrin receptors,mutations which increased affinity did not appear to substantially alterthe manner in which the antibody interacted with the receptor.

[0203] The ability of the variants to inhibit the adhesion of M21 humanmelanoma cells expressing the α_(v)β₃ receptor to fibrinogen wasexamined. Inhibition of the adhesion of 4×10⁴ M21 cells to fibrinogen bythe LM609 grafted antibody variants was performed as describedpreviously (Leavesley et al., J. Cell Biol. 117:1101-1107 (1992)).Similar to the ligand competition studies with purified fibrinogen andα_(v)β₃ receptor, higher affinity variants were generally more effectiveat preventing cell adhesion than was LM609 grafted antibody (FIG. 11).FIG. 11 shows inhibition of M21 human melanoma cell adhesion tofibrinogen. Cells and various concentrations of LM609 grafted antibodyFab (closed triangles), S102 (open circles), G102 (closed circles), orC37 (open triangles) were added to 96 well cell culture plates which hadbeen coated with 10 μg/ml fibrinogen. After incubating for 35 min at 37°C., unbound cells were removed by washing and adherent cells werequantitated by crystal violet staining.

[0204] Although intact LM609 grafted antibody Ig inhibits cell adhesion,the phage expressed Fab did not affect cell adhesion at concentrationsas high as 1 mg/ml (FIG. 11, closed triangles). Clone C37, isolated fromthe combinatorial library and displaying ˜90-fold greater affinity thanLM609 grafted antibody Fab, inhibited cell adhesion completely (FIG. 11,open triangles). Variant G102 had a moderately higher affinity (2.2-foldenhanced) and also inhibited cell adhesion, though less effectively thanC37 (FIG. 11, closed circles). Surprisingly, clone S102 (FIG. 11, opencircles), which had a 4.6-fold higher affinity than LM609 graftedantibody, was ineffective at inhibiting cell adhesion, suggesting thatclones G102 and S102 interact with the α_(v)β₃ receptor differently.

[0205] These results show that combining single amino acid mutationsthat result in LM609 grafted antibodies exhibiting higher bindingaffinity to α_(v)β₃ allows the identification of high affinity LM609grafted antibody mutants having greater than 90-fold higher bindingaffinity than the parent LM609 grafted antibody.

[0206] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 100 351 base pairs nucleic acid both linear CDS 1..351 1 CAG GTG CAGCTG GTG GAG TCT GGG GGA GGC GTT GTG CAG CCT GGA AGG 48 Gln Val Gln LeuVal Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 TCC CTG AGACTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGT AGC TAT 96 Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 GAC ATG TCT TGGGTT CGC CAG GCT CCG GGC AAG GGT CTG GAG TGG GTC 144 Asp Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 GCA AAA GTT AGT AGTGGT GGT GGT AGC ACC TAC TAT TTA GAC ACT GTG 192 Ala Lys Val Ser Ser GlyGly Gly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 CAG GGC CGA TTC ACC ATCTCC AGA GAC AAT AGT AAG AAC ACC CTA TAC 240 Gln Gly Arg Phe Thr Ile SerArg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 CTG CAA ATG AAC TCT CTGAGA GCC GAG GAC ACA GCC GTG TAT TAC TGT 288 Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 GCA AGA CAT AAC TAC GGC AGTTTT GCT TAC TGG GGC CAA GGG ACT ACA 336 Ala Arg His Asn Tyr Gly Ser PheAla Tyr Trp Gly Gln Gly Thr Thr 100 105 110 GTG ACT GTT TCT AGT 351 ValThr Val Ser Ser 115 117 amino acids amino acid linear protein 2 Gln ValGln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 AspMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 AlaLys Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 GlnGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg His Asn Tyr Gly Ser Phe Ala Tyr Trp Gly Gln Gly Thr Thr 100 105110 Val Thr Val Ser Ser 115 321 base pairs nucleic acid both linear CDS1..321 3 GAG ATT GTG CTA ACT CAG TCT CCA GCC ACC CTG TCT CTC AGC CCA GGA48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 510 15 GAA AGG GCG ACT CTT TCC TGC CAG GCC AGC CAA AGT ATT AGC AAC CAC 96Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Ser Ile Ser Asn His 20 25 30CTA CAC TGG TAT CAA CAA AGG CCT GGT CAA GCC CCA AGG CTT CTC ATC 144 LeuHis Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 AAGTAT CGT TCC CAG TCC ATC TCT GGG ATC CCC GCC AGG TTC AGT GGC 192 Lys TyrArg Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 AGT GGATCA GGG ACA GAT TTC ACC CTC ACT ATC TCC AGT CTG GAG CCT 240 Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 GAA GATTTT GCA GTC TAT TAC TGT CAA CAG AGT GGC AGC TGG CCT CAC 288 Glu Asp PheAla Val Tyr Tyr Cys Gln Gln Ser Gly Ser Trp Pro His 85 90 95 ACG TTC GGAGGG GGG ACC AAG GTG GAA ATT AAG 321 Thr Phe Gly Gly Gly Thr Lys Val GluIle Lys 100 105 107 amino acids amino acid linear protein 4 Glu Ile ValLeu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu ArgAla Thr Leu Ser Cys Gln Ala Ser Gln Ser Ile Ser Asn His 20 25 30 Leu HisTrp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys TyrArg Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 GluAsp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Gly Ser Trp Pro His 85 90 95 ThrPhe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 351 base pairs nucleicacid both linear CDS 1..351 5 GAA GTG CAG CTG GTG GAG TCT GGG GGA GGCTTA GTG AAG CCT GGA AGG 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Lys Pro Gly Arg 1 5 10 15 TCC CTG AGA CTC TCC TGT GCA GCC TCT GGATTC GCT TTC AGT AGC TAT 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheAla Phe Ser Ser Tyr 20 25 30 GAC ATG TCT TGG GTT CGC CAG ATT CCG GAG AAGAGG CTG GAG TGG GTC 144 Asp Met Ser Trp Val Arg Gln Ile Pro Glu Lys ArgLeu Glu Trp Val 35 40 45 GCA AAA GTT AGT AGT GGT GGT GGT AGC ACC TAC TATTTA GAC ACT GTG 192 Ala Lys Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr LeuAsp Thr Val 50 55 60 CAG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCC AAG AACACC CTA TAC 240 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn ThrLeu Tyr 65 70 75 80 CTG CAA ATG AGC AGT CTG AAC TCT GAG GAC ACA GCC ATGTAT TAC TGT 288 Leu Gln Met Ser Ser Leu Asn Ser Glu Asp Thr Ala Met TyrTyr Cys 85 90 95 GCA AGA CAT AAC TAC GGC AGT TTT GCT TAC TGG GGC CAA GGGACT CTG 336 Ala Arg His Asn Tyr Gly Ser Phe Ala Tyr Trp Gly Gln Gly ThrLeu 100 105 110 GTC ACT GTC TCT GCA 351 Val Thr Val Ser Ala 115 117amino acids amino acid linear protein 6 Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg GlnIle Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Lys Val Ser Ser Gly GlyGly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Gln Gly Arg Phe Thr Ile SerArg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser LeuAsn Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Asn Tyr GlySer Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala115 321 base pairs nucleic acid both linear CDS 1..321 7 GAT ATT GTG CTAACT CAG TCT CCA GCC ACC CTG TCT GTG ACA CCA GGA 48 Asp Ile Val Leu ThrGln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 GAT AGC GTC AGTCTT TCC TGC CAG GCC AGC CAA AGT ATT AGC AAC CAC 96 Asp Ser Val Ser LeuSer Cys Gln Ala Ser Gln Ser Ile Ser Asn His 20 25 30 CTA CAC TGG TAT CAACAA AAA TCA CAT GAG TCT CCA AGG CTT CTC ATC 144 Leu His Trp Tyr Gln GlnLys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 AAG TAT CGT TCC CAG TCCATC TCT GGG ATC CCC TCC AGG TTC AGT GGC 192 Lys Tyr Arg Ser Gln Ser IleSer Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 AGT GGA TCA GGG ACA GAT TTCGCT CTC AGT ATC AAC AGT GTG GAG ACT 240 Ser Gly Ser Gly Thr Asp Phe AlaLeu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 GAA GAT TTT GGA ATG TAT TTCTGT CAA CAG AGT GGC AGC TGG CCT CAC 288 Glu Asp Phe Gly Met Tyr Phe CysGln Gln Ser Gly Ser Trp Pro His 85 90 95 ACG TTC GGA GGG GGG ACC AAG CTGGAA ATT AAG 321 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 107amino acids amino acid linear protein 8 Asp Ile Val Leu Thr Gln Ser ProAla Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser CysGln Ala Ser Gln Ser Ile Ser Asn His 20 25 30 Leu His Trp Tyr Gln Gln LysSer His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Arg Ser Gln Ser IleSer Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp PheAla Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Met TyrPhe Cys Gln Gln Ser Gly Ser Trp Pro His 85 90 95 Thr Phe Gly Gly Gly ThrLys Leu Glu Ile Lys 100 105 84 base pairs nucleic acid both linear 9CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTTGTGCAGC CTGGAAGGTC CCTGAGACTC 60TCCTGTGCAG CCTCTGGATT CACC 84 84 base pairs nucleic acid both linear 10AACTTTTGCG ACCCACTCCA GACCCTTGCC CGGAGCCTGG CGAACCCAAG ACATGTCATA 60GCTACTGAAG GTGAATCCAG AGGC 84 88 base pairs nucleic acid both linear 11GTGGGTCGCA AAAGTTAGTA GTGGTGGTGG TAGCACCTAC TATTTAGACA CTGTGCAGGG 60CCGATTCACC ATCTCCAGAG ACAATAGT 88 81 base pairs nucleic acid both linear12 TGCACAGTAA TACACGGCTG TGTCCTCGGC TCTCAGAGAG TTCATTTGCA GGTATAGGGT 60GTTCTTACTA TTGTCTCTGG A 81 75 base pairs nucleic acid both linear 13GTGTATTACT GTGCAAGACA TAACTACGGC AGTTTTGCTT ACTGGGGCCA AGGGACTACA 60GTGACTGTTT CTAGT 75 87 base pairs nucleic acid both linear 14 GAGATTGTGCTAACTCAGTC TCCAGCCACC CTGTCTCTCA GCCCAGGAGA AAGGGCGACT 60 CTTTCCTGCCAGGCCAGCCA AAGTATT 87 72 base pairs nucleic acid both linear 15GATGAGAAGC CTTGGGGCTT GACCAGGCCT TTGTTGATAC CAGTGTAGGT GGTTGCTAAT 60ACTTTGGCTG GC 72 84 base pairs nucleic acid both linear 16 CCAAGGCTTCTCATCWASTA TCGTTCCCAG TCCATCTCTG GGATCCCCGC CAGGTTCAGT 60 GGCAGTGGATCAGGGACAGA TTTC 84 81 base pairs nucleic acid both linear 17 GCTGCCACTCTGTTGACAGW AATAGACTGC AAAATCTTCA GGCTCCAGAC TGGAGATAGT 60 GAGGGTGAAATCTGTCCCTG A 81 57 base pairs nucleic acid both linear 18 CAACAGAGTGGCAGCTGGCC TCACACGTTC GGAGGGGGGA CCAAGGTGGA AATTAAG 57 36 base pairsnucleic acid single linear 19 GCCCAACCAG CCATGGCCGA TATTGTGCTA ACTCAG 3620 base pairs nucleic acid single linear 20 ACAGTTGGTG CAGCATCAGC 20 36base pairs nucleic acid single linear 21 ACCCCTGTGG CAAAAGCCGAAGTGCAGCTG GTGGAG 36 20 base pairs nucleic acid both linear 22GATGGGGGTG TCGTTTTGGC 20 87 base pairs nucleic acid both linear 23GAGATTGTGC TAACTCAGTC TCCAGCCACC CTGTCTCTCA GCCCAGGAGA AAGGGCGACT 60CTTTCCTGCC AGGCCAGCCA AAGTATT 87 75 base pairs nucleic acid both linear24 TTAGATGAGA AGCCTTGGGG CTTGACCAGG CCTTTGTTGA TACCAGTGTA GGTGGTTGCT 60AATACTTTGG CTGGC 75 84 base pairs nucleic acid both linear 25 CCAAGGCTTCTCATCTAATA TCGTTCCCAG TCCATCTCTG GGATCCCCGC CAGGTTCAGT 60 GGCAGTGGATCAGGGACAGA TTTC 84 81 base pairs nucleic acid both linear 26 GCTGCCACTCTGTTGACAGT AATAGACTGC AAAATCTTCA GGCTCCAGAC TGGAGATAGT 60 GAGGGTGAAATCTGTCCCTG A 81 57 base pairs nucleic acid both linear 27 CAACAGAGTGGCAGCTGGCC TCACACGTTC GGAGGGGGGA CCAAGGTGGA AATTAAG 57 10 amino acidsamino acid linear 28 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 1 5 10 22base pairs nucleic acid both linear 29 GCTACTGAAG GCGAATCCAG AG 22 23base pairs nucleic acid single linear misc_feature 11..13 /note= “”NNN“represents a codon specifying any amino acid other than Lys.” 30GGGAACGATA NNNGATGAGA AGC 23 321 base pairs nucleic acid single linearCDS 1..321 31 GAG ATT GTG CTA ACT CAG TCT CCA GCC ACC CTG TCT CTC AGCCCA GGA 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser ProGly 1 5 10 15 GAA AGG GCG ACT CTT TCC TGC CAG GCC AGC CAA AGT ATT AGCAAC CAC 96 Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Ser Ile Ser AsnHis 20 25 30 CTA CAC TGG TAT CAA CAA AGG CCT GGT CAA GCC CCA AGG CTT CTCATC 144 Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile35 40 45 MKK TAT CGT TCC CAG TCC ATC TCT GGG ATC CCC GCC AGG TTC AGT GGC192 Xaa Tyr Arg Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 5055 60 AGT GGA TCA GGG ACA GAT TTC ACC CTC ACT ATC TCC AGT CTG GAG CCT240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 6570 75 80 GAA GAT TTT GCA GTC TAT TAC TGT CAA CAG AGT GGC AGC TGG CCT CAC288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Gly Ser Trp Pro His 8590 95 ACG TTC GGA GGG GGG ACC AAG GTG GAA ATT AAG 321 Thr Phe Gly GlyGly Thr Lys Val Glu Ile Lys 100 105 107 amino acids amino acid linearprotein 32 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser ProGly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Ser Ile SerAsn His 20 25 30 Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg LeuLeu Ile 35 40 45 Xaa Tyr Arg Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Gly SerTrp Pro His 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10530 base pairs nucleic acid both linear CDS 1..30 33 GGA TTC ACC TTC AGTAGC TAT GAC ATG TCT 30 Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser 1 5 10 10amino acids amino acid linear protein 34 Gly Phe Thr Phe Ser Ser Tyr AspMet Ser 1 5 10 30 base pairs nucleic acid both linear CDS 1..30 35 TGGGTC GCA AAA GTT AGT AGT GGT GGT GGT 30 Trp Val Ala Lys Val Ser Ser GlyGly Gly 1 5 10 10 amino acids amino acid linear protein 36 Trp Val AlaLys Val Ser Ser Gly Gly Gly 1 5 10 30 base pairs nucleic acid bothlinear CDS 1..30 37 AGC ACC TAC TAT TTA GAC ACT GTG CAG GGC 30 Ser ThrTyr Tyr Leu Asp Thr Val Gln Gly 1 5 10 10 amino acids amino acid linearprotein 38 Ser Thr Tyr Tyr Leu Asp Thr Val Gln Gly 1 5 10 30 base pairsnucleic acid both linear CDS 1..30 39 GCA AGA CAT AAC TAC GGC AGT TTTGCT TAC 30 Ala Arg His Asn Tyr Gly Ser Phe Ala Tyr 1 5 10 10 amino acidsamino acid linear protein 40 Ala Arg His Asn Tyr Gly Ser Phe Ala Tyr 1 510 39 base pairs nucleic acid both linear CDS 1..39 41 CAG GCC AGC CAAAGT ATT AGC AAC CAC CTA CAC TGG TAT 39 Gln Ala Ser Gln Ser Ile Ser AsnHis Leu His Trp Tyr 1 5 10 13 amino acids amino acid linear protein 42Gln Ala Ser Gln Ser Ile Ser Asn His Leu His Trp Tyr 1 5 10 33 base pairsnucleic acid both linear CDS 1..33 43 CTT CTC ATC CGT TAT CGT TCC CAGTCC ATC TCT 33 Leu Leu Ile Arg Tyr Arg Ser Gln Ser Ile Ser 1 5 10 11amino acids amino acid linear protein 44 Leu Leu Ile Arg Tyr Arg Ser GlnSer Ile Ser 1 5 10 27 base pairs nucleic acid both linear CDS 1..27 45CAA CAG AGT GGC AGC TGG CCT CAC ACG 27 Gln Gln Ser Gly Ser Trp Pro HisThr 1 5 9 amino acids amino acid linear protein 46 Gln Gln Ser Gly SerTrp Pro His Thr 1 5 30 base pairs nucleic acid both linear CDS 1..30 47GGA ACT ACC TTC AGT AGC TAT GAC ATG TCT 30 Gly Thr Thr Phe Ser Ser TyrAsp Met Ser 1 5 10 10 amino acids amino acid linear protein 48 Gly ThrThr Phe Ser Ser Tyr Asp Met Ser 1 5 10 30 base pairs nucleic acid bothlinear CDS 1..30 49 GGA TTC ACC TGG AGT AGC TAT GAC ATG TCT 30 Gly PheThr Trp Ser Ser Tyr Asp Met Ser 1 5 10 10 amino acids amino acid linearprotein 50 Gly Phe Thr Trp Ser Ser Tyr Asp Met Ser 1 5 10 30 base pairsnucleic acid both linear CDS 1..30 51 GGA TTC ACC TTC CTG AGC TAT GACATG TCT 30 Gly Phe Thr Phe Leu Ser Tyr Asp Met Ser 1 5 10 10 amino acidsamino acid linear protein 52 Gly Phe Thr Phe Leu Ser Tyr Asp Met Ser 1 510 30 base pairs nucleic acid both linear CDS 1..30 53 TGG GTC GCA AAAGTT AAA AGT GGT GGT GGT 30 Trp Val Ala Lys Val Lys Ser Gly Gly Gly 1 510 10 amino acids amino acid linear protein 54 Trp Val Ala Lys Val LysSer Gly Gly Gly 1 5 10 30 base pairs nucleic acid both linear CDS 1..3055 AGC ACC TAC TAT CCT GAC ACT GTG CAG GGC 30 Ser Thr Tyr Tyr Pro AspThr Val Gln Gly 1 5 10 10 amino acids amino acid linear protein 56 SerThr Tyr Tyr Pro Asp Thr Val Gln Gly 1 5 10 30 base pairs nucleic acidboth linear CDS 1..30 57 AGC ACC TAC TAT TTA GAC ACT GTG GAG GGC 30 SerThr Tyr Tyr Leu Asp Thr Val Glu Gly 1 5 10 10 amino acids amino acidlinear protein 58 Ser Thr Tyr Tyr Leu Asp Thr Val Glu Gly 1 5 10 30 basepairs nucleic acid both linear CDS 1..30 59 GCA AGA CAT AAC CAT GGC AGTTTT GCT TAC 30 Ala Arg His Asn His Gly Ser Phe Ala Tyr 1 5 10 10 aminoacids amino acid linear protein 60 Ala Arg His Asn His Gly Ser Phe AlaTyr 1 5 10 30 base pairs nucleic acid both linear CDS 1..30 61 GCA AGACAT AAC TAC GGC AGT TAT GCT TAC 30 Ala Arg His Asn Tyr Gly Ser Tyr AlaTyr 1 5 10 10 amino acids amino acid linear protein 62 Ala Arg His AsnTyr Gly Ser Tyr Ala Tyr 1 5 10 30 base pairs nucleic acid both linearCDS 1..30 63 GCA AGA CAT AAC TAC GGC AGT TTT GAT TAC 30 Ala Arg His AsnTyr Gly Ser Phe Asp Tyr 1 5 10 10 amino acids amino acid linear protein64 Ala Arg His Asn Tyr Gly Ser Phe Asp Tyr 1 5 10 30 base pairs nucleicacid both linear CDS 1..30 65 GCA AGA CAT AAC TAC GGC AGT TTT TAT TAC 30Ala Arg His Asn Tyr Gly Ser Phe Tyr Tyr 1 5 10 10 amino acids amino acidlinear protein 66 Ala Arg His Asn Tyr Gly Ser Phe Tyr Tyr 1 5 10 30 basepairs nucleic acid both linear CDS 1..30 67 GCA AGA CAT AAC TAC GGC AGTTTT GCT TCT 30 Ala Arg His Asn Tyr Gly Ser Phe Ala Ser 1 5 10 10 aminoacids amino acid linear protein 68 Ala Arg His Asn Tyr Gly Ser Phe AlaSer 1 5 10 30 base pairs nucleic acid both linear CDS 1..30 69 GCA AGACAT AAC TAC GGC AGT TTT GCT ACT 30 Ala Arg His Asn Tyr Gly Ser Phe AlaThr 1 5 10 10 amino acids amino acid linear protein 70 Ala Arg His AsnTyr Gly Ser Phe Ala Thr 1 5 10 30 base pairs nucleic acid both linearCDS 1..30 71 GCA AGA CAT AAC TAC GGC AGT TTT GCT GAT 30 Ala Arg His AsnTyr Gly Ser Phe Ala Asp 1 5 10 10 amino acids amino acid linear protein72 Ala Arg His Asn Tyr Gly Ser Phe Ala Asp 1 5 10 30 base pairs nucleicacid both linear CDS 1..30 73 GCA AGA CAT AAC TAC GGC AGT TTT GCT GAG 30Ala Arg His Asn Tyr Gly Ser Phe Ala Glu 1 5 10 10 amino acids amino acidlinear protein 74 Ala Arg His Asn Tyr Gly Ser Phe Ala Glu 1 5 10 30 basepairs nucleic acid both linear CDS 1..30 75 GCA AGA CAT AAC TAC GGC AGTTTT GCT ATG 30 Ala Arg His Asn Tyr Gly Ser Phe Ala Met 1 5 10 10 aminoacids amino acid linear protein 76 Ala Arg His Asn Tyr Gly Ser Phe AlaMet 1 5 10 30 base pairs nucleic acid both linear CDS 1..30 77 GCA AGACAT AAC TAC GGC AGT TTT GCT GGG 30 Ala Arg His Asn Tyr Gly Ser Phe AlaGly 1 5 10 10 amino acids amino acid linear protein 78 Ala Arg His AsnTyr Gly Ser Phe Ala Gly 1 5 10 30 base pairs nucleic acid both linearCDS 1..30 79 GCA AGA CAT AAC TAC GGC AGT TTT GCT GCT 30 Ala Arg His AsnTyr Gly Ser Phe Ala Ala 1 5 10 10 amino acids amino acid linear protein80 Ala Arg His Asn Tyr Gly Ser Phe Ala Ala 1 5 10 39 base pairs nucleicacid both linear CDS 1..39 81 CAG GCC AGC CAA AGT ATT AGC AAC TTT CTACAC TGG TAT 39 Gln Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr 1 510 13 amino acids amino acid linear protein 82 Gln Ala Ser Gln Ser IleSer Asn Phe Leu His Trp Tyr 1 5 10 33 base pairs nucleic acid bothlinear CDS 1..33 83 CTT CTC ATC CGT TAT TCT TCC CAG TCC ATC TCT 33 LeuLeu Ile Arg Tyr Ser Ser Gln Ser Ile Ser 1 5 10 11 amino acids amino acidlinear protein 84 Leu Leu Ile Arg Tyr Ser Ser Gln Ser Ile Ser 1 5 10 27base pairs nucleic acid both linear CDS 1..27 85 CAA CAG AGT AAT AGC TGGCCT CAC ACG 27 Gln Gln Ser Asn Ser Trp Pro His Thr 1 5 9 amino acidsamino acid linear protein 86 Gln Gln Ser Asn Ser Trp Pro His Thr 1 5 27base pairs nucleic acid double linear CDS 1..27 87 CAA CAG AGT ACT AGCTGG CCT CAC ACT 27 Gln Gln Ser Thr Ser Trp Pro His Thr 1 5 9 amino acidsamino acid linear protein 88 Gln Gln Ser Thr Ser Trp Pro His Thr 1 5 27base pairs nucleic acid both linear CDS 1..27 89 CAA CAG AGT GGC AGC TGGCCT CTG ACG 27 Gln Gln Ser Gly Ser Trp Pro Leu Thr 1 5 9 amino acidsamino acid linear protein 90 Gln Gln Ser Gly Ser Trp Pro Leu Thr 1 5 27base pairs nucleic acid both linear CDS 1..27 91 CAA CAG AGT GGC AGC TGGCCT CAG ACG 27 Gln Gln Ser Gly Ser Trp Pro Gln Thr 1 5 9 amino acidsamino acid linear protein 92 Gln Gln Ser Gly Ser Trp Pro Gln Thr 1 5 30base pairs nucleic acid both linear CDS 1..30 93 GCA AGA CAT AAC CAT GGCAGT TTT GCT TCT 30 Ala Arg His Asn His Gly Ser Phe Ala Ser 1 5 10 10amino acids amino acid linear protein 94 Ala Arg His Asn His Gly Ser PheAla Ser 1 5 10 30 base pairs nucleic acid both linear CDS 1..30 95 GCAAGA CAT AAC CAT GGC AGT TTT TAT TCT 30 Ala Arg His Asn His Gly Ser PheTyr Ser 1 5 10 10 amino acids amino acid linear protein 96 Ala Arg HisAsn His Gly Ser Phe Tyr Ser 1 5 10 30 base pairs nucleic acid bothlinear CDS 1..30 97 GCA AGA CAT AAC TAC GGC AGT TTT TAT GAG 30 Ala ArgHis Asn Tyr Gly Ser Phe Tyr Glu 1 5 10 10 amino acids amino acid linearprotein 98 Ala Arg His Asn Tyr Gly Ser Phe Tyr Glu 1 5 10 30 base pairsnucleic acid both linear CDS 1..30 99 GCA AGA CAT AAC TAC GGC AGT TTTTAT TCT 30 Ala Arg His Asn Tyr Gly Ser Phe Tyr Ser 1 5 10 10 amino acidsamino acid linear protein 100 Ala Arg His Asn Tyr Gly Ser Phe Tyr Ser 15 10

What is claimed is:
 1. A Vitaxin antibody exhibiting selective bindingaffinity to α_(v)β₃ comprising at least one Vitaxin heavy chainpolypeptide comprising substantially the same variable region amino acidsequence as that shown in FIG. 1A (SEQ ID NO:2) and at least one Vitaxinlight chain polypeptide comprising substantially the same variableregion amino acid sequence as that shown in FIG. 1B (SEQ ID NO:4) or afunctional fragment thereof.
 2. The Vitaxin antibody of claim 1, whereinsaid functional fragment is selected from the group consisting of Fv,Fab, F(ab)₂ and scFV.
 3. A nucleic acid encoding a Vitaxin heavy chainpolypeptide comprising substantially the same Vitaxin heavy chainvariable region nucleotide sequences as that shown in FIG. 1A (SEQ IDNO:1) or a fragment thereof.
 4. The nucleic acid of claim 3, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same nucleotide sequence as the variable region of said Vitaxinheavy chain polypeptide (SEQ ID NO:1).
 5. The nucleic acid of claim 3,wherein said fragment further comprises a nucleic acid encodingsubstantially the same nucleotide sequence as a CDR of said Vitaxinheavy chain polypeptide.
 6. A nucleic acid encoding a Vitaxin lightchain polypeptide comprising substantially the same Vitaxin light chainvariable region nucleotide sequences as that shown in FIG. 1B (SEQ IDNO:3) or a fragment thereof.
 7. The nucleic acid of claim 6, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same nucleotide sequence as the variable region of said Vitaxinlight chain polypeptide (SEQ ID NO:3).
 8. The nucleic acid of claim 6,wherein said fragment further comprises a nucleic acid encodingsubstantially the same nucleotide sequence as a CDR of said Vitaxinlight chain polypeptide.
 9. A nucleic acid encoding a Vitaxin heavychain polypeptide comprising a nucleotide sequence encodingsubstantially the same Vitaxin heavy chain variable region amino acidsequence as that shown in FIG. 1A (SEQ ID NO:2) or fragment thereof. 10.The nucleic acid of claim 9, wherein said fragment further comprises anucleic acid encoding substantially the same heavy chain variable regionamino acid sequence of said Vitaxin heavy chain amino acid sequence (SEQID NO:2).
 11. The nucleic acid of claim 9, wherein said fragment furthercomprises a nucleic acid encoding substantially the same heavy chain CDRamino acid sequence of said Vitaxin heavy chain amino acid sequence. 12.A nucleic acid encoding a Vitaxin light chain polypeptide comprising anucleotide sequence encoding substantially the same Vitaxin light chainvariable region amino acid sequence as that shown in FIG. 1B (SEQ IDNO:4) or fragment thereof.
 13. The nucleic acid of claim 12, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same light chain variable region amino acid sequence of said Vitaxinlight chain amino acid sequence (SEQ ID NO:4).
 14. The nucleic acid ofclaim 12, wherein said fragment further comprises a nucleic acidencoding substantially the same light chain CDR amino acid sequence ofsaid Vitaxin light chain amino acid sequence.
 15. A Vitaxin heavy chainpolypeptide comprising substantially the same variable region amino acidsequence as that shown in FIG. 1A (SEQ ID NO:2) or functional fragmentthereof.
 16. The Vitaxin heavy chain polypeptide of claim 15, whereinsaid functional fragment comprises a variable chain polypeptide or a CDRpolypeptide.
 17. A Vitaxin light chain polypeptide comprisingsubstantially the same variable region amino acid sequence as that shownin FIG. 1B (SEQ ID NO:4) or a functional fragment thereof.
 18. TheVitaxin light chain polypeptide of claim 17, wherein said functionalfragment comprises a variable chain polypeptide or a CDR polypeptide.19. A LM609 grafted antibody exhibiting selective binding affinity toα_(v)β₃ comprising at least one LM609 grafted heavy chain polypeptidecomprising substantially the same variable region amino acid sequence asthat shown in FIG. 1A (SEQ ID NO:2) and at least one LM609 grafted lightchain polypeptide comprising substantially the same variable regionamino acid sequence as that shown in FIG. 7 (SEQ ID NO:32) or afunctional fragment thereof.
 20. The LM609 grafted antibody of claim 19,wherein said functional fragment is selected from the group consistingof Fv, Fab, F(ab)₂ and scFV.
 21. A nucleic acid encoding a LM609 graftedheavy chain polypeptide comprising substantially the same LM609 graftedheavy chain variable region nucleotide sequences as that shown in FIG.1A (SEQ ID NO:1) or a fragment thereof.
 22. The nucleic acid of claim21, wherein said fragment further comprises a nucleic acid encodingsubstantially the same nucleotide sequence as the variable region ofsaid LM609 grafted heavy chain polypeptide (SEQ ID NO:1).
 23. Thenucleic acid of claim 21, wherein said fragment further comprises anucleic acid encoding substantially the same nucleotide sequence as aCDR of said LM609 grafted heavy chain polypeptide.
 24. A nucleic acidencoding a LM609 grafted light chain polypeptide comprisingsubstantially the same LM609 grafted light chain variable regionnucleotide sequences as that shown in FIG. 7 (SEQ ID NO:31) or afragment thereof.
 25. The nucleic acid of claim 24, wherein saidfragment further comprises a nucleic acid encoding substantially thesame nucleotide sequence as the variable region of said LM609 graftedlight chain polypeptide (SEQ ID NO:31).
 26. The nucleic acid of claim24, wherein said fragment further comprises a nucleic acid encodingsubstantially the same nucleotide sequence as a CDR of said LM609grafted light chain polypeptide.
 27. A nucleic acid encoding a LM609grafted antibody heavy chain polypeptide comprising a nucleotidesequence encoding substantially the same LM609 grafted heavy chainvariable region amino acid sequence as that shown in FIG. 1A (SEQ IDNO:2) or fragment thereof.
 28. The nucleic acid of claim 27, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same heavy chain variable region amino acid sequence of said LM609grafted heavy chain amino acid sequence (SEQ ID NO:2).
 29. The nucleicacid of claim 27, wherein said fragment further comprises a nucleic acidencoding substantially the same heavy chain CDR amino acid sequence ofsaid LM609 grafted heavy chain amino acid sequence.
 30. A nucleic acidencoding a LM609 grafted antibody light chain polypeptide comprising anucleotide sequence encoding substantially the same LM609 grafted lightchain variable region amino acid sequence as that shown in FIG. 7 (SEQID NO:32) or fragment thereof.
 31. The nucleic acid of claim 30, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same light chain variable region amino acid sequence of said Vitaxinlight chain amino acid sequence (SEQ ID NO:32).
 32. The nucleic acid ofclaim 30, wherein said fragment further comprises a nucleic acidencoding substantially the same light chain CDR amino acid sequence ofsaid Vitaxin light chain amino acid sequence.
 33. A LM609 grafted heavychain polypeptide comprising substantially the same variable regionamino acid sequence as that shown in FIG. 1A (SEQ ID NO:2) or functionalfragment thereof.
 34. The LM609 grafted heavy chain polypeptide of claim33, wherein said functional fragment comprises a variable chainpolypeptide or a CDR polypeptide.
 35. A LM609 grafted light chainpolypeptide comprising substantially the same variable region amino acidsequence as that shown in FIG. 7 (SEQ ID NO:32) or a functional fragmentthereof.
 36. The LM609 grafted light chain polypeptide of claim 35,wherein said functional fragment comprises a variable chain polypeptideor a CDR polypeptide.
 37. A nucleic acid encoding a heavy chainpolypeptide for monoclonal antibody LM609 comprising substantially thesame heavy chain variable region nucleotide sequence as that shown inFIG. 2A (SEQ ID NO:5) or a fragment thereof.
 38. The nucleic acid ofclaim 37, wherein said fragment further comprises a nucleic acidencoding substantially the same nucleotide sequence as the variableregion of said heavy chain polypeptide (SEQ ID NO:5).
 39. The nucleicacid of claim 37, wherein said fragment further comprises a nucleic acidencoding substantially the same nucleotide sequence as a CDR of saidheavy chain polypeptide.
 40. A nucleic acid encoding a light chainpolypeptide for monoclonal antibody LM609 comprising substantially thesame light chain variable region nucleotide sequence as that shown inFIG. 2B (SEQ ID NO:7) or a fragment thereof.
 41. The nucleic acid ofclaim 40, wherein said fragment further comprises a nucleic acidencoding substantially the same nucleotide sequence as the variableregion of said light chain polypeptide (SEQ ID NO:7).
 42. The nucleicacid of claim 40, wherein said fragment further comprises a nucleic acidencoding substantially the same nucleotide sequence as a CDR of saidlight chain polypeptide.
 43. A nucleic acid encoding a heavy chainpolypeptide for monoclonal antibody LM609 comprising a nucleotidesequence encoding substantially the same heavy chain variable domainamino acid sequence of monoclonal antibody LM609 as that shown in FIG.2A (SEQ ID NO:6) or fragment thereof.
 44. The nucleic acid of claim 43,wherein said fragment further comprises a nucleic acid encodingsubstantially the same heavy chain variable region amino acid sequenceof said monoclonal antibody LM609 (SEQ ID NO:6).
 45. The nucleic acid ofclaim 43, wherein said fragment further comprises a nucleic acidencoding substantially the same heavy chain CDR amino acid sequence assaid monoclonal antibody LM609.
 46. A nucleic acid encoding a heavychain polypeptide for monoclonal antibody LM609 comprising a nucleotidesequence encoding substantially the same light chain amino acid sequenceof monoclonal antibody LM609 as that shown in FIG. 2B (SEQ ID NO:8) orfragment thereof.
 47. The nucleic acid of claim 46, wherein saidfragment further comprises a nucleic acid encoding substantially thesame light chain variable region amino acid sequence of said monoclonalantibody LM609 (SEQ ID NO:8).
 48. The nucleic acid of claim 46, whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same light chain CDR amino acid sequence as said monoclonal antibodyLM609.
 49. A method of inhibiting a function of α_(v)β₃ comprisingcontacting α_(v)β₃ with Vitaxin or a functional fragment thereof underconditions which allow binding of Vitaxin to α_(v)β₃.
 50. The method ofclaim 49, wherein said functional fragment is selected from the groupconsisting of Fv, Fab, F(ab) ₂ and scFV.
 51. The method of claim 49,wherein said function of α_(v)β₃ is binding of α_(v)β₃ to a ligand. 52.The method of claim 49, wherein said function of α_(v)β₃ is integrinmediated signal transduction.
 53. A method of treating anα_(v)β₃-mediated disease comprising administering an effective amount ofVitaxin or a functional fragment thereof under conditions which allowbinding to α_(v)β₃.
 54. The method of claim 53, wherein said functionalfragment is selected from the group consisting of Fv, Fab, F(ab) ₂ andscFV.
 55. The method of claim 53, wherein said α_(v)β₃-mediated diseaseis angiogenesis or restenosis.
 56. An enhanced LM609 grafted antibodyexhibiting selective binding affinity to α_(v)β₃, or a functionalfragment thereof, comprising at least one amino acid substitution in oneor more CDRs of a LM609 grafted heavy chain variable region polypeptideor a LM609 grafted light chain variable region polypeptide, wherein theα_(v)β₃ binding affinity of said enhanced LM609 grafted antibody ismaintained.
 57. The enhanced LM609 grafted antibody of claim 56, whereinsaid α_(v)β₃ binding affinity of said LM609 grafted antibody isenhanced.
 58. The enhanced LM609 grafted antibody of claim 56, whereinsaid functional fragment is selected from the group consisting of Fv,Fab, F(ab)₂ and scFV.
 59. The enhanced LM609 grafted antibody of claim56, wherein said CDR having at least one amino acid substitution isselected from the group consisting of V_(H) CDR1, V_(H) CDR2, V_(H)CDR3, V_(L) CDR1, V_(L) CDR2 and V_(L) CDR3.
 60. The enhanced LM609grafted antibody of claim 59, wherein said V_(H) CDR1 is selected fromthe group consisting of the CDRs referenced as SEQ ID NO:48, SEQ IDNO:50 and SEQ ID NO:52.
 61. The enhanced LM609 grafted antibody of claim59, wherein said V_(H) CDR2 is selected from the group consisting of theCDRs referenced as SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:58.
 62. Theenhanced LM609 grafted antibody of claim 59, wherein said V_(H) CDR3 isselected from the group consisting of the CDRs referenced as SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:94, SEQ ID NO:96; SEQ ID NO:98 and SEQ ID NO:100. 63.The enhanced LM609 grafted antibody of claim 59, wherein said V_(L) CDR1is the CDR referenced as SEQ ID NO:82.
 64. The enhanced LM609 graftedantibody of claim 59, wherein said V_(L) CDR2 is the CDR referenced asSEQ ID NO:84.
 65. The enhanced LM609 grafted antibody of claim 59,wherein said V_(L) CDR3 is selected from the group consisting of theCDRs referenced as SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 and SEQ IDNO:92.
 66. The enhanced LM609 grafted antibody of claim 56, wherein saidenhanced LM609 grafted antibody comprises at least one amino acidsubstitution in two or more CDRs of a LM609 grafted heavy chain variableregion polypeptide or a LM609 grafted light chain variable regionpolypeptide.
 67. The enhanced LM609 grafted antibody of claims 66,wherein said functional fragment is selected from the group consistingof Fv, Fab, F(ab)₂ and scFV.
 68. The enhanced LM609 grafted antibody ofclaim 66, wherein said CDR having at least one amino acid substitutionis selected from the group consisting of V_(H) CDR1, V_(H) CDR2, V_(H)CDR3, V_(L) CDR1, V_(L) CDR2 and V_(L) CDR3.
 69. The enhanced LM609grafted antibody of claim 68, wherein said enhanced LM609 graftedantibody comprises the combination of CDRs selected from the groupconsisting of: the V_(L) CDR1 referenced as SEQ ID NO:82 and the V_(H)CDR3 referenced as SEQ ID NO:68; the V_(L) CDR1 referenced as SEQ IDNO:82, the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3referenced as SEQ ID NO:68; the V_(L) CDR1 referenced as SEQ ID NO:82,the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3 referencedas SEQ ID NO:72; the V_(L) CDR1 referenced as SEQ ID NO:82, the V_(H)CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3 referenced as SEQ IDNO:70; the V_(L) CDR1 referenced as SEQ ID NO:82 and the V_(H) CDR3referenced as SEQ ID NO:72; the V_(L) CDR3 referenced as SEQ ID NO:86,the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3 referencedas SEQ ID NO:68; the V_(L) CDR3 referenced as SEQ ID NO:90 and V_(H)CDR3 referenced as SEQ ID NO:68; and the V_(L) CDR3 referenced as SEQ IDNO:90, the V_(H) CDR2 referenced as SEQ ID NO:56 and V_(H) CDR3referenced as SEQ ID NO:68.
 70. The enhanced LM609 grafted antibody ofclaim 66, wherein at least one of said CDRs has two or more amino acidsubstitutions.
 71. The enhanced LM609 grafted antibody of claims 70,wherein said functional fragment is selected from the group consistingof Fv, Fab, F(ab)₂ and scFV.
 72. The enhanced LM609 grafted antibody ofclaim 70, wherein said CDR having at least one amino acid substitutionis selected from the group consisting of V_(H) CDR1, V_(H) CDR2, V_(H)CDR3, V_(L) CDR1, V_(L) CDR2 and V_(L) CDR3.
 73. The enhanced LM609grafted antibody of claim 72, wherein said enhanced LM609 graftedantibody comprises the combination of CDRs selected from the groupconsisting of: the V_(L) CDR1 referenced as SEQ ID NO:82, the V_(H) CDR2referenced as SEQ ID NO:56 and the V_(H) CDR3 referenced as SEQ IDNO:94; the V_(L) CDR3 referenced as SEQ ID NO:90, the V_(H) CDR2referenced as SEQ ID NO:56 and the V_(H) CDR3 referenced as SEQ IDNO:94; the V_(L) CDR3 referenced as SEQ ID NO:90, the V_(H) CDR2referenced as SEQ ID NO:56 and the V_(H) CDR3 referenced as SEQ IDNO:96; the V_(L) CDR3 referenced as SEQ ID NO:90 and the V_(H) CDR3referenced as SEQ ID NO:94; the V_(L) CDR3 referenced as SEQ ID NO:90and the V_(H) CDR3 referenced as SEQ ID NO:98; and the V_(L) CDR3referenced as SEQ ID NO:90, the V_(H) CDR2 referenced as SEQ ID NO:56and the V_(H) CDR3 referenced as SEQ ID NO:100.
 74. A high affinityLM609 grafted antibody exhibiting selective binding affinity to α_(v)β₃,or a functional fragment thereof, comprising at least one amino acidsubstitution in one or more CDRs of a LM609 grafted heavy chain variableregion polypeptide or a LM609 grafted light chain variable regionpolypeptide, wherein the α_(v)β₃ binding affinity of said high affinityLM609 grafted antibody is enhanced.
 75. The high affinity LM609 graftedantibody of claim 74, wherein said functional fragment is selected fromthe group consisting of Fv, Fab, F(ab)₂ and scFV.
 76. The high affinityLM609 grafted antibody of claim 74, wherein said CDR having at least oneamino acid substitution is selected from the group consisting of V_(H)CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2 and V_(L) CDR3. 77.The high affinity LM609 grafted antibody of claim 76, wherein said highaffinity LM609 grafted antibody comprises the combination of CDRsselected from the group consisting of: the V_(L) CDR1 referenced as SEQID NO:82 and the V_(H) CDR3 referenced as SEQ ID NO:68; the V_(L) CDR1referenced as SEQ ID NO:82, the V_(H) CDR2 referenced as SEQ ID NO:56and the V_(H) CDR3 referenced as SEQ ID NO:68; the V_(L) CDR1 referencedas SEQ ID NO:82, the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H)CDR3 referenced as SEQ ID NO:72; the V_(L) CDR1 referenced as SEQ IDNO:82, the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3referenced as SEQ ID NO:70; the V_(L) CDR1 referenced as SEQ ID NO:82and the V_(H) CDR3 referenced as SEQ ID NO:72; the V_(L) CDR3 referencedas SEQ ID NO:86, the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H)CDR3 referenced as SEQ ID NO:68; the V_(L) CDR3 referenced as SEQ IDNO:90, the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3referenced as SEQ ID NO:94; the V_(L) CDR3 referenced as SEQ ID NO:90and V_(H) CDR3 referenced as SEQ ID NO:68; the V_(L) CDR3 referenced asSEQ ID NO:90, the V_(H) CDR2 referenced as SEQ ID NO:56 and V_(H) CDR3referenced as SEQ ID NO:68; the V_(L) CDR1 referenced as SEQ ID NO:82,the V_(H) CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3 referencedas SEQ ID NO:94; the V_(L) CDR3 referenced as SEQ ID NO:90, the V_(H)CDR2 referenced as SEQ ID NO:56 and the V_(H) CDR3 referenced as SEQ IDNO:96; the V_(L) CDR3 referenced as SEQ ID NO:90 and the V_(H) CDR3referenced as SEQ ID NO:94; the V_(L) CDR3 referenced as SEQ ID NO:90and the V_(H) CDR3 referenced as SEQ ID NO:98; and the V_(L) CDR3referenced as SEQ ID NO:90, the V_(H) CDR2 referenced as SEQ ID NO:56and the V_(H) CDR3 referenced as SEQ ID NO:100.
 78. A nucleic acidencoding an enhanced LM609 grafted antibody, or a functional fragmentthereof, exhibiting selective binding affinity to α_(v)β₃ comprising atleast one amino acid substitution in one or more CDRs of a LM609 graftedheavy chain variable region polypeptide or a LM609 grafted light chainvariable region polypeptide, wherein the α_(v)β₃ binding affinity ofsaid enhanced LM609 grafted antibody is maintained or enhanced.
 79. Anucleic acid encoding a high affinity LM609 grafted antibody, or afunctional fragment thereof, exhibiting selective binding affinity toα_(v)β₃ comprising at least one amino acid substitution in one or moreCDRs of a LM609 grafted heavy chain variable region polypeptide or aLM609 grafted light chain variable region polypeptide, wherein theα_(v)β₃ binding affinity of said high affinity LM609 grafted antibody isenhanced.